The Mechanism of Amino Acid Activation: the Work of Mahlon Hoagland

Enzymatic Carboxyl Activation of Amino Acids(Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. (1956) J. Biol. Chem. 218, 345–358)Mahlon Bush Hoagland was born in Boston, Massachusetts in 1921. He attended Harvard University and graduated in 1943. Knowing that he wanted to be a surgeon, Hoagland then enrolled at Harvard Medical School. However, he was diagnosed with tuberculosis, and his poor health prevented him from becoming a surgeon when he received his M.D. in 1948. Instead, he accepted a research position at Massachusetts General Hospital. In 1953, he became a postdoctoral fellow with Journal of Biological Chemistry (JBC) Classic author Fritz Lipmann (1) at Huntington Laboratories (also at Massachusetts General Hospital), and a year later, he moved to an adjoining laboratory to work on protein synthesis with JBC Classic author Paul Zamecnik (2).Open in a separate windowMahlon HoaglandInspired by Lipmann''s insights into acyl activation mechanisms, Hoagland used a cell-free system created by Zamecnik that carried out net peptide bond formation using ¹⁴C-amino acids (3) to uncover the mechanism of amino acid activation. As reported in the JBC Classic reprinted here, he isolated an enzyme fraction that, in the presence of ATP and amino acids, catalyzed the first step in protein synthesis: the formation of aminoacyl adenylates or activated amino acids. Using data from analysis of this fraction, Hoagland presented a scheme for amino acid activation in his Classic paper.A few years later, Zamecnik and Hoagland discovered a molecule that is essential for protein synthesis: tRNA. This discovery is the subject of the Zamecnik Classic (2).After the discovery of tRNA, Hoagland spent the next year (1957–1958) at Cambridge University''s Cavendish laboratories working with Francis Crick. During that year he traveled to France to visit the Institute Pasteur in Paris. Experiments begun at the Institute would, by 1960, lead to the discovery of messenger RNA (mRNA).When he returned to the United States, Hoagland was appointed associate professor of microbiology at Harvard Medical School. He remained there until 1967 when he accepted a position as professor at Dartmouth Medical School. In 1970, he became the director of the Worcester Foundation for Experimental Biology, a Massachusetts research institute founded by his father. He retired in 1985 and currently lives in Thetford, Vermont.Hoagland has received several awards and honors in recognition of his contributions to science. These include the 1976 Franklin Medal, the 1982 and 1996 Book Awards from the American Medical Writers Association, and membership in the American Academy of Arts and Sciences and the National Academy of Sciences.     

The characterization of restriction endonucleases: the work of Hamilton Smith

Purification of the HhaII Restriction Endonuclease from an Overproducer Escherichia coli Clone(Kelly, S., Kaddurah-Daouk, R., and Smith, H. O. (1985) J. Biol. Chem. 260, 15339–15344)Catalytic Properties of the HhaII Restriction Endonuclease(Kaddurah-Daouk, R., Cho, P., and Smith, H. O. (1985) J. Biol. Chem. 260, 15345–15351)Hamilton Othanel Smith was born in 1931 in New York City. In 1937, he and his family moved to Champaign-Urbana, Illinois, because his father had joined the faculty of the department of education at the University of Illinois. As a boy, Smith was interested in chemistry, electricity, and electronics, and he spent many hours with his brother in their basement laboratory, which was stocked with supplies purchased from their paper route earnings. Smith attended a small college preparatory school called the University Laboratory High School and graduated in 3 years largely due to his science teacher who allowed him to complete chemistry and physics during the summer.Open in a separate windowHamilton O. SmithAfter finishing high school, Smith enrolled at the University of Illinois, majoring in mathematics. During his sophomore year, his brother showed him a book on mathematical modeling of central nervous system circuits by Nicolas Rashevsky. This caught his interest, and after transferring to the University of California, Berkeley, Smith immersed himself in courses in cell physiology, biochemistry, and biology. A guest lecture by Journal of Biological Chemistry (JBC) Classic author George Wald (1) describing his studies of retinal biochemistry soon converted Smith into a devoted student of visual physiology and eventually motivated him to apply to medical school.In 1952, Smith began his studies at the Johns Hopkins University Medical School. He received his M.D. 4 years later and went to Barnes Hospital in St. Louis for a medical internship. However, in 1957, Smith was called up in the Doctor Draft and joined the U.S. Navy. He finished his Navy service in 1959 and moved to Detroit to begin a medical residency training at the Henry Ford Hospital. There he became interested in bacteriophage and decided that this would be the focus of his research.So, in 1962, Smith began his research career with Myron Levine in the department of human genetics at the University of Michigan in Ann Arbor. He and Levine carried out a series of studies demonstrating the sequential action of the phage P22 C-genes, which controlled lysogenization. They also discovered the gene controlling prophage attachment, now known as the int gene, and carried out a study of defective transducing particles formed after induction of int mutant prophage.In 1967, Smith joined the faculty of Johns Hopkins University as an assistant professor of microbiology and continued his bacteriophage research. A year later, working with Thomas J. Kelly, Jr. and Kent W. Wilcox, Smith isolated and characterized the first Type II restriction endonuclease (HindII) from Haemophilus influenzae and determined the sequence of its cleavage site (2, 3). In recognition of this discovery, he was awarded the 1978 Nobel Prize in Physiology or Medicine with Werner Arber and Daniel Nathans.These studies led to Smith''s subsequent research on DNA methylases and nucleases in H. influenzae. The two JBC Classics reprinted here detail Smith''s efforts to discover the rules governing sequence recognition in the Type II restriction endonuclease HhaII via x-ray crystallography. To facilitate these studies, Smith and his colleagues engineered a two-plasmid system in Escherichia coli that overproduced HhaII on induction with isopropylthiogalactoside (IPTG). The first paper describes the induction characteristics of the two-plasmid overproducer clone and purification of the endonuclease. The second paper, published back-to-back with the first, details the catalytic properties of the endonuclease. Smith used two methods to follow the reactions: 1) gel electrophoretic analysis of nicked circular and linear DNA products, and 2) release of ³²P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. Smith''s two-plasmid system eventually allowed him to obtain crystals of the HhaII endonuclease with a heptanucleotide DNA duplex (4).Smith served on the faculty at Johns Hopkins for 30 years before retiring as American Cancer Society Distinguished Research Professor Emeritus of Molecular Biology and Genetics in 1998. In 1993, he accepted an appointment to the scientific advisory council of The Institute for Genomic Research, which led to his collaboration with J. Craig Venter in the sequencing of H. influenzae by whole genome shotgun sequencing and assembly. Five years later, Smith joined Celera Genomics, where he was senior director of DNA Resources and aided in the sequencing of the Drosophila and human genomes. In 2005, he co-founded Synthetic Genomics, an off-shoot of Celera. He also serves as scientific director of the Synthetic Biology & Biological Energy Groups at the J. Craig Venter Institute. In addition to the Nobel Prize, Smith has received several honors including election to the National Academy of Sciences in 1980.¹     

From Nuclear Science to Bacterial Cytochromes: the Work of Martin D. Kamen

Structural Bases for Function in Cytochromes c. An Interpretation of Comparative X-ray and Biochemical Data (Salemme, F. R., Kraut, J., and Kamen, M. D. (1973) J. Biol. Chem. 248, 7701–7716) Martin David Kamen (1913–2002) was born in Toronto, Canada, but grew up in Chicago. He enrolled at the University of Chicago in 1930, intending to study English. However, the Great Depression took a toll on his family''s finances, and his father suggested he switch his major to chemistry in order to make a living after graduating. By his junior year, Kamen was hooked on chemistry. He graduated in 1933 with honors in physical chemistry, working with William Draper Harkins to determine ammonia gas emission spectra excited by electrodeless discharge. He remained in Harkins'' lab for graduate school, earning his doctorate in physical chemistry in 1936 for an article on neutron scattering (1) that was accepted as his dissertation.Open in a separate windowMartin D. KamenBecause economic conditions were still bleak, Kamen followed the suggestion of one of his mentors, David Gans, and applied for a research post with Nobel laureate Ernest O. Lawrence, who had developed the cyclotron at the radiation laboratory in Berkeley, California. Kamen used his savings to move to Berkeley and worked at the laboratory without pay for 6 months before Lawrence offered him a staff position as a chemist. In addition to troubleshooting the cyclotrons and preparing samples of radioisotopes, Kamen performed numerous photosynthetic studies with Samuel Ruben, using carbon-11. Because carbon-11 had a half-life of only 21 min, Lawrence assigned Kamen and Ruben the task of finding carbon-14. The pair succeeded by bombarding graphite in the cyclotron, producing carbon-14, which had a 5730-year half-life (2).Kamen and Ruben planned to use their discovery to create a company that would construct and operate several cyclotrons dedicated to carbon-14 production and expand on the laboratory''s radioisotope program. However, the war intervened, and all non-war-related research at Berkeley was halted. Kamen was assigned to head a program studying the separation of uranium isotopes for the Manhattan Project. But, unexpectedly in 1944, he was declared a security risk and dismissed from the lab. A few years later, Kamen was called before the House Un-American Activities Committee, being wrongly linked to an espionage ring working for the USSR. Subsequently, the State Department refused to issue Kamen a passport, and the Chicago Tribune named him as a suspected spy. During the next decade, he fought recurring rumors and accusations that he had leaked atomic bomb secrets. Eventually, he won a libel suit against the Chicago Tribune, and the State Department reinstated his passport.In 1945, Kamen moved to the Mallinckrodt Institute of Radiology at the Washington University School of Medicine where he supervised cyclotron production of radioisotopes for medical research. His own research interests gradually shifted away from nuclear physics and radiochemistry to biochemistry, and he began several collaborations involving the use of radioisotopic tracers in biological and biomedical research.Kamen also initiated a series of experiments using carbon-14 to study photosynthesis in bacteria. This resulted in a number of important discoveries, including hydrogen photoevolution (3) and nitrogen fixation (4) in Rhodospirillum rubrum. While working with the bacteria, Kamen and Leo Vernon discovered that R. rubrum contained a c-type cytochrome (5), which they later named “cytochrome c₂.”Twenty years after it was discovered, the structure of cytochrome c₂ was solved (6). By comparing this structure with the recently solved structure of eukaryotic mitochondrial cytochrome c (7), Kamen and his colleagues were able to deduce information about the structural, functional, and evolutionary relationships in the cytochromes c. This is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here.Despite the fact that both eukaryotic cytochrome c and cytochrome c₂ serve analogous functions in their respective physiological electron transport chains, i.e. they both transport electrons to the terminal and most oxidizing electron carrier of each system, Kamen was able to find several differences between the molecules. For example, he noted that cytochrome c₂ has a more positive electrochemical potential and does not exhibit the large oxidation state-dependent conformational change characteristic of mitochondrial cytochrome c. Open in a separate windowKamen continued to study other bacterial cytochromes, showing that at least 12 subgroups of the cytochromes c exist. This resulted in new perspectives on potential variations in structure and function of the heme group in relation to protein.In 1957 Kamen moved to Brandeis University to help establish the graduate department of biochemistry, and in 1961 he joined the University of California, San Diego chemistry department to help found their new campus. He remained there until 1975, when he became director of the Chemical-Biological Development Laboratory at the University of Southern California. Kamen continued to teach into his eighties, being one of six faculty members of the Oregon Institute of Science and Medicine.Kamen received numerous awards and honors for his contributions to science, including the American Chemical Society''s Award for Applications of Nuclear Chemistry (1963), the American Society of Plant Physiologists'' Charles F. Kettering Research Award (1968), the American Society of Biological Chemists'' Merck Award (1982), the John Scott Medal of the City of Philadelphia (1988), the World Cultural Council''s Einstein Award (1990), and the U.S. Department of Energy''s Enrico Fermi Award (1996). He was a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the American Philosophical Society. ¹     

Signaling through Tyr985 of Leptin Receptor as an Age/Diet-Dependent Switch in the Regulation of Energy Balance

Leptin regulates energy homeostasis through central activation of multiple signaling pathways mediated by Ob-Rb, the long form of leptin receptor. Leptin resistance underlies the pathogenic development of obesity, which is closely associated with environmental factors. To further understand the physiological function of leptin signaling mechanisms, we generated a knock-in line of mice (Y985F) expressing a mutant Ob-Rb with a phenylalanine substitution for Tyr⁹⁸⁵, one of the three intracellular tyrosines that mediate leptin''s signaling actions. Surprisingly, whereas young homozygous Y985F animals were slightly leaner, they exhibit adult-onset or diet-induced obesity. Importantly, both age-dependent and diet-induced deterioration of energy balance was paralleled with pronounced leptin resistance, which was largely attributable to attenuation of leptin-responsive hypothalamic STAT3 activation as well as prominently elevated expression of hypothalamic SOCS3, a key negative regulator of leptin signaling. Thus, these results unmask distinct binary roles for Try⁹⁸⁵-mediated signaling in energy metabolism, acting as an age/diet-dependent regulatory switch to counteract age-associated or diet-induced obesity.As a component of the metabolic syndrome, obesity is closely associated with increased risk for the development of type 2 diabetes and cardiovascular disorders (16). Arising from a chronic imbalance between energy intake and expenditure, the pathogenic progression of obesity is attributable to the complex interactions between genetic factors and environmental influences. In mammals, energy balance is maintained through multiple homeostatic mechanisms that operate coordinately in response to hormonal and nutritional cues. Leptin is an adipose-secreted hormone (43) that plays a pivotal role in the regulation of energy metabolism. Acting through its active-form receptor Ob-Rb in distinct classes of leptin-responsive neurons (11, 14, 34), leptin activates multiple signaling pathways in the hypothalamus to regulate food intake and energy expenditure. Mice with deficiency in leptin (ob/ob) or its functional receptor (db/db) develop morbid obesity, hyperphagia, and diabetes (20). Impaired leptin responsiveness, i.e., leptin resistance (33), is a key characteristic of the metabolic defects that are responsible for disrupted energy control, presumably underlying the pathogenic development of human obesity (29). Although diminished leptin signaling has been found to occur in association with aging (21, 39) or feeding of a high-fat diet (HFD) (17, 18), the exact physiological mechanisms linking the environmental factors to the impairment in leptin-mediated regulation of energy metabolism remain largely elusive.Leptin binds to Ob-Rb and elicits an array of subsequent intracellular signaling cascades (7, 22) via Jak2 phosphorylation. The mouse Ob-Rb comprises three cytoplasmic tyrosine residues, Tyr⁹⁸⁵, Tyr¹⁰⁷⁷, and Tyr¹¹³⁸, which are known to be phosphorylated and mediate leptin''s physiological functions (22, 26). The phosphorylated Tyr¹¹³⁸ is thought to recruit STAT3 (1), thereby activating the JAK2-STAT3 pathway, which has been shown to play an important role in the control of energy balance (2), whereas our recent investigation has demonstrated crucial actions in vivo for Ob-Rb tyrosine-dependent as well as tyrosine-independent mechanisms in the regulation of energy and glucose homeostasis (26). Our earlier studies in vitro as well as observations from other laboratories have also documented that phosphorylation at Tyr⁹⁸⁵ leads to recruitment of SH2-containing protein tyrosine phosphatase 2 (SHP2) (28, 41) and activation of extracellular signal-regulated kinase (ERK) (9). On the other hand, phosphorylated Tyr⁹⁸⁵ has been postulated to serve as a docking site for SOCS3, thereby exerting an antagonizing effect on Tyr¹¹³⁸-mediated STAT3 activation (8). Consistent with this, a leptin-activated autoinhibitory action in vivo has recently been suggested for Tyr⁹⁸⁵ in the l/l mice expressing a mutant leptin receptor where Tyr⁹⁸⁵ was replaced with leucine (10). However, whereas elevated hypothalamic expression of SOCS3 has been reported to occur in aged rodents (36) or in mice with diet-induced obesity (18, 42), it has yet to be understood whether Ob-Rb Tyr⁹⁸⁵-mediated mechanisms are physiologically connected to altered SOCS3 expression, particularly in the face of aging or high dietary fat intake. Moreover, direct in vivo evidence also has been lacking with respect to whether there exist potential interplays between Ob-Rb Tyr⁹⁸⁵ signaling and other Ob-Rb tyrosine-dependent mechanisms, which act to influence the homeostatic control of energy balance.To gain further insight into the roles of Ob-Rb intracellular tyrosine phosphorylation in mediating leptin''s physiological functions in vivo, we previously generated two lines of mice expressing mutant leptin receptors with phenylalanine substitution for all three tyrosines or for Tyr¹¹³⁸ alone, revealing the metabolic contribution of both tyrosine-dependent and -independent actions in energy homeostasis (26). Here we investigated the physiological consequences of abrogation of signaling through Ob-Rb Tyr⁹⁸⁵ via characterization of the knock-in mice generated by introducing a phenylalanine substitution mutation at this site. We examined the impact of deficiency in Tyr⁹⁸⁵-mediated signaling upon the susceptibility of mice to age-associated and diet-induced energy imbalance, attempting to explore the potential mechanistic links between leptin resistance and environmental influences such as aging and overnutrition.     

The Role of Binding Energy in Catalysis: the Work of William P. Jencks

Two Functional Domains of Coenzyme A Activate Catalysis by Coenzyme A Transferase. Pantetheine and Adenosine 3′-Phosphate 5′-Diphosphate (Fierke, C. A., and Jencks, W. P. (1986) J. Biol. Chem. 261, 7603–7606)William Platt Jencks (1927–2007) was born in Bar Harbor, Maine. He became interested in chemistry when he received a chemistry set for Christmas in 1934. He immediately carried out one of the experiments described in the instructions, the addition of dilute acid to a sulfide salt to produce H₂S. The experiment was so successful that his house had to be evacuated due to the smell of rotten eggs. According to Jencks, “My family and I did not find it necessary to replicate this experiment” (1).Open in a separate windowWilliam P. JencksJencks enrolled at Harvard College, intending to study chemistry. However, after taking a first year course in chemistry that “described a large number of chemical reactions, one after the other, with no indication of what was interesting about any of them” (1), he switched his major to English. Despite this change in the direction of his studies, Jencks ended up entering Harvard Medical School after his junior year because he wasn''t sure what else to do.After completing his first year of medical school, Jencks spent a summer at the Marine Biological Laboratory in Woods Hole, taking courses and doing research on lobster shell pigments with Journal of Biological Chemistry (JBC) Classic author George Wald (2). He received his M.D. in 1951 and then interned at Peter Bent Brigham Hospital in Boston. However, after a while, Jencks found medicine to be “a very broad field in which it would be difficult to obtain definitive answers to fundamental problems” (1). Wald suggested Jencks try doing research at Massachusetts General Hospital with Nobel laureate Fritz Lipmann (who was featured in a previous JBC Classic (3)). Jencks ended up spending 2 years with Lipmann, studying coenzyme A transferase, which led to his longtime interest in the physical organic chemistry of acyl transfer reactions. After leaving Massachusetts General Hospital, Jencks spent a year doing postdoctoral studies at Harvard University with Nobel laureate Robert Woodward before joining the faculty at Brandeis University in 1957, serving as assistant, associate, and then full professor of biochemistry. He retired in 1996 as professor emeritus of biochemistry.During his 39 years at Brandeis University, Jencks studied the mechanisms by which enzymes facilitate chemical reactions of molecules that are not otherwise inclined to react at a useful rate.The JBC Classic reprinted here looks at the noncovalent interactions between succinyl-CoA 3-ketoacid coenzyme A transferase and coenzyme A. In the paper, Jencks and Carol A. Fierke used a small coenzyme A analog, methylmercaptopropionate, to show that noncovalent interactions between the enzyme and the side chain of CoA are responsible for the reaction rate increase brought about by the enzyme. They report that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of substrate destabilization and rate acceleration, whereas the interaction with the 3′-phospho-ADP1 moiety provides binding energy that overcomes this destabilization and permits significant binding of acyl-CoA substrates to the enzyme. This paper helped to illuminate a striking example of the role of binding energy in catalysis.Jencks received many honors and awards for his contributions to science, including memberships in the National Academy of Sciences (1971) and the American Philosophical Society (1995) and foreign membership in the Royal Society. He also received the 1962 American Chemical Society (ACS) Award in Biological Chemistry, the 1993 American Society of Biological Chemists Award, the 1995 ACS James Flack Norris Award in Physical Organic Chemistry, and the 1996 ACS Repligen Award for Chemistry of Biological Processes.¹     

Yasutomi Nishizuka's Discovery of Protein Kinase C

Studies on a Cyclic Nucleotide-independent Protein Kinase and Its Proenzyme in Mammalian Tissues. I. Purification and Characterization of an Active Enzyme from Bovine Cerebellum (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603–7609)Direct Activation of Calcium-activated, Phospholipid-dependent Protein Kinase by Tumor-promoting Phorbol Esters (Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847–7851)Yasutomi Nishizuka (1932–2004) was born in Ashiya-city, Japan. He attended Kyoto University and obtained his M.D. in 1957 and his Ph.D. in 1962, working with Journal of Biological Chemistry (JBC) Classic author Osamu Hayaishi (1). He then spent a year as a postdoctoral fellow at Rockefeller University with Fritz Lipmann (also featured in a JBC Classic (2)) before returning to Kyoto University to resume work with Hayaishi. During this time, Nishizuka studied the biosynthesis of nicotinamide adenine dinucleotide (NAD), the involvement of GTP in ribosomal protein translation, and ADP-ribosylation by diphtheria toxin.Open in a separate windowYasutomi NishizukaIn 1969, Nishizuka accepted the position of full professor and head of the department of biochemistry at the Kobe University School of Medicine. There, Nishizuka became interested in the role of protein kinases in the regulation of cell functions. This led to his discovery of a novel protein kinase, which he published in the first paper reprinted here as the JBC Classic. As Nishizuka reported in that paper, he and his colleagues partially purified the kinase from bovine cerebellum. They found that the enzyme was capable of phosphorylating histone and protamine and that it probably was produced from its precursor protein by a limited proteolytic reaction. The detailed properties of the proenzyme and its conversion to active protein kinase were reported in a subsequent JBC paper (3). Nishizuka named this new enzyme “protein kinase C (PKC).”A paper published by Nishizuka two years later in the JBC (4) showed that PKC was activated without limited proteolysis by a membrane-associated factor in the presence of a low concentration of Ca²⁺. In 1980, he published another paper in the JBC (5) showing that the membrane-associated factor was diacylglycerol, which suggested that the lipid could be a novel second messenger generated by receptor-stimulated phosphoinositide hydrolysis. Nishizuka validated this idea by showing that treating platelets with a combination of a Ca²⁺ ionophore and membrane-permeant short chain diacylglycerol mimicked stimulation by the aggregating agent thrombin (6). This discovery was a major advance in the understanding of cell signaling.Nishizuka and his colleagues then discovered that PKC is the biological target of tumor-promoting phorbol esters. At that time, it was well known that croton oil augmented carcinogenesis when it was applied at weekly intervals to the skin of mice in conjunction with a very dilute solution of benz[a]pyrene in acetone. The oil contained phorbol ester, a powerful tumor promoter, and caused a wide variety of cellular responses that were similar to those seen with hormones. Nishizuka speculated that the phorbol ester was producing diacylglycerol to activate PKC. However, upon further investigation, he realized that the phorbol ester contained a diacylglycerol-like structure and thus might activate PKC directly. In a series of experiments, published in the second JBC Classic reprinted here, Nishizuka was able to show that the phorbol ester activated PKC directly. This discovery showed that PKC was important for cell proliferation and cancer. It also established the use of phorbol esters as crucial tools for the manipulation of PKC activation in intact cells, eventually allowing the elucidation of the wide range of cellular processes regulated by this enzyme.This research laid the foundation for an enormous number of studies on the complex PKC family, many of them from Nishizuka''s group.In 1975, Nishizuka became president of the University of Kobe, a position that he held until 2001. He received numerous awards and honors for his research, including the Gairdner Foundation International Award (1988), the Alfred P. Sloan Jr. Prize (1988), the Japan Order of Culture (1988), the Albert Lasker Basic Medical Research Award (1989), the Kyoto Prize (1992), the Wolf Prize in Medicine (1995), the Jimenez Diaz Award (1995), and the Schering Prize (1995). He also served as a foreign member and honorary fellow of various academies, including the National Academies of Science, the Royal Society, l''Academie des Sciences, die Deutsche Akademie der Naturforscher Leopoldina, le Real Academia de Ciencias, the Asiatic Society, and the Japan Academy.¹     

Spectroscopic Studies of Carboxypeptidase: the Work of Bert L. Vallee

The Role of Zinc in Carboxypeptidase (Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1960) J. Biol. Chem. 235, 64–69)Metallocarboxypeptidases (Coleman, J. E., and Vallee, B. L. (1960) J. Biol. Chem. 235, 390–395)Bert Lester Vallee (1919–2010) was born in Germany and grew up in Luxembourg. He received his B.S. in 1938 from the University of Bern in Switzerland, after which he came to the United States as the first (and only) fellow of the League of United Nations International Student Service. He was admitted to New York University and worked with Richard Courant, receiving his M.D. from the New York University College of Medicine in 1943.Open in a separate windowBert L. ValleeDuring World War II, Vallee was assigned to the joint Harvard Medical School-MIT blood preservation project, directed by Journal of Biological Chemistry (JBC) Classic authors Edwin Cohn (1) and John Edsall (2). This experience shaped Vallee''s future career in biochemistry and biophysics. At MIT, he became interested in the metabolism of iron and other metals such as zinc and copper. Little was known about this topic at the time, and he recognized the potential of spectroscopy, particularly emission and arc spectroscopy, for the detection of metals in biological systems. He decided to focus his research on the subject and was awarded a National Research Council Fellowship to pursue these studies in the physics and biology departments at MIT under the direction of John R. Loofbourow and George R. Harrison.In 1954, Vallee joined the faculty of Harvard Medical School where he established the Biophysics Research Laboratory. He was named assistant professor of medicine in 1956 and rose swiftly through the ranks to become the Paul C. Cabot Professor of Biological Chemistry in 1965 and the Edgar M. Bronfman Distinguished Senior Professor in 1980 (a title he held until his death in 2010).At Harvard, Vallee continued the work that he initiated at MIT, focusing on the design and construction of new spectrochemical instruments for use in biology. He built a flame spectrometer to detect and quantify sodium, potassium, magnesium, and calcium in biological samples. At that time, these elements could not be measured accurately in physiological concentrations. This early spectrometer became the prototype for later instruments capable of monitoring these elements in clinical samples.Vallee''s laboratory soon became a world center for the analysis of trace metals in biological samples. He continued his efforts to define, develop, and evaluate new spectroscopic flame sources for the excitation and spectral emission of atoms and to make biological spectroscopy an intrinsic part of modern biological and medical science. Because of his work on the role of metals in biological systems, many consider him to be the “father of metallobiochemistry.”Vallee''s own research centered on the identification of zinc in various metalloproteins and enzymes. One of the many zinc proteins he studied was carboxypeptidase. Vallee carried out careful and extensive mechanistic studies of the enzyme using spectroscopy, stopped-flow kinetics, and chemical modification. He was able not only to elucidate its reaction mechanism but also to provide structural information on the enzyme, including particular roles of specific amino acids. When the x-ray structure of carboxypeptidase was ultimately deduced, Vallee''s results proved to be remarkably accurate.The two JBC Classics reprinted here were both published in 1960 and contain some of Vallee''s early observations on carboxypeptidase. In the first Classic, Vallee and several of his colleagues, including JBC Classic author Hans Neurath (3), examined the roll of zinc in carboxypeptidase. Vallee and Neurath had previously determined that each carboxypeptidase molecule contained one atom of zinc and that the zinc was necessary for enzymatic activity (4). They followed up these initial observations by showing that enzymatic activity lost by zinc dialysis is exactly proportional to the amount of zinc removed. They also discovered that the loss of activity could be reversed by the addition of zinc and several other ions, including Cr³⁺, Ni²⁺, Co²⁺, Fe²⁺, and Mn²⁺.Later that year, Vallee and Joseph E. Coleman published the second JBC Classic, which explores the relationship between pH and the restoration of activity by the addition of metal ions to carboxypeptidase. Vallee discovered that the degree to which activity is restored is a critical function of pH. Because pH could influence enzymatic activity either by affecting the binding of the metal to the apoenzyme or by influencing the rate of catalysis, Vallee and Coleman attempted to separate these two effects by exposing the enzyme to the metal ions at a given pH but assaying it under standard conditions at pH 7.5. They determined that the restoration of activity was a direct function of the binding of metal to the apoprotein. They also learned that the apoenzyme had at least two binding sites for metals but that only one of the sites was essential for activity.Vallee also studied the zinc-containing alcohol dehydrogenase and the role the enzyme plays in alcohol metabolism and alcohol addiction. He showed that genetics are an important determinant of alcoholism, and his work has led to clinical trials of drugs for the treatment of this disease, including daidzin, which he isolated from the kudzu plant.Since Vallee started working on zinc-containing proteins, scientists have found that up to 10% of the human proteome may be composed of zinc proteins, and as of April 2007, there are nearly 400 x-ray and NMR structures of zinc proteins available.Vallee was widely recognized for his scientific achievements. He received many awards, including the Warner-Chilcott Award (1969), the Linderstrom-Lang Medal (1980), the Willard Gibbs Medal from the American Chemical Society (1981), and the William C. Rose Award from the American Society for Biochemistry and Molecular Biology (1982). He also was elected to the National Academy of Sciences and the American Academy of Arts and Sciences. Wanting to give back to science, Vallee and his wife established the Vallee Foundation to foster originality, creativity, and leadership in science. The Foundation funds honorary Vallee Professorships, which allow accomplished scientists to explore new areas and to establish close interactions with other successful senior investigators that might lead to new knowledge.     

Mycobacterial Glycophosphoinositides: the Work of Clinton E. Ballou

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family of Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046–3056)Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acylation of the Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 2975–2984)Clinton Edward Edgerton Ballou was born in King Hill, Idaho in 1923. After graduating from high school, he enrolled in a premed program at Boise Junior College, but his interests quickly turned to chemistry after he dissected a poorly embalmed cat in a comparative anatomy course. During his sophomore year, Ballou transferred to Oregon State College in Corvallis where he became involved in two research projects: the first during his sophomore year synthesizing new antimalarial drugs with Bert Christensen, and the second during his senior year studying the guinea pig “antistiffness factor” with Willem van Wagtendonk.Open in a separate windowClinton E. BallouThe military draft was in effect as Ballou entered his last year of college so he decided to join the U. S. Navy after graduating in 1944. He was discharged 2 years later and decided to apply for graduate study in biochemistry with Karl Paul Link at the University of Wisconsin-Madison. As detailed in a previous Journal of Biological Chemistry (JBC) Classic (1), Link''s research centered on blood anticoagulants, and when Ballou arrived in his laboratory in 1946, the primary focus was the structure-function relationship of coumarin anticoagulants. Ballou was immediately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarol because the acetylated intermediate was degraded in the alkali conditions used for deacetylation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Ballou decided to study a variety of synthetic compounds to try to understand the structural basis for alkali sensitivity. This research formed the core of his doctoral dissertation, and his exposure to carbohydrate chemistry influenced the direction of his career.After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L. Hirst in the Department of Chemistry at the University of Edinburgh. There he studied the structure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to work with Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at the University of California, Berkeley. Ballou explained his choice: “I was attracted to Fischer in part because of his research on phosphorylated sugars but also because during graduate school I had drawn heavily on the published works of his father, Emil Fischer. I guess the idea of being associated with the son of Emil Fischer just seemed ‘real cool’ to me” (2).The 1950s was a time of active research on biosynthetic pathways involving short chain phosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou undertook the syntheses of several such metabolic intermediates, including d-glyceric acid 2-phosphate, d-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate, and d-erythrose 4-phosphate. He also became interested in inositol chemistry as a result of studies on the cyclitols in sugar pine heartwood.In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about setting up an independent research program. He decided to work on inositol-containing phospholipids and was able to synthesize and characterize d-myoinositol 1-phosphate. He also spent several years isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide. This culminated in his discovery of d-myoinositol l,4,5-trisphosphate or IP₃. More information on Ballou''s studies of these phosphoinositides can be found in his JBC Reflections (2).Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at the National Center for Scientific Research (CNRS) in France studying the glycophosphoinositides of mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experiments to establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositol ring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structures of the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4–6). Ballou and his postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intact dimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBC Classics reprinted here.In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyze the biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reported that the major products are a family of three dimannophosphoinositides (A, B, and C) that differ in the number of fatty acyl groups they contain (four, three, and two, respectively). On the basis of their results, they proposed that guanosine diphosphate mannose acts as the sugar donor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside (dimannophosphoinositide C), which is then acylated in a two-step process to first yield dimannophosphoinositide B and then dimannophosphoinositide A.In the second JBC Classic, Ballou and Brennan provide further evidence for their proposed biosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fatty acids into the dimannophosphoinositides. They showed that label from [¹⁴C]palmityl-CoA is incorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After a short incubation period, this molecule is converted to dimannophosphoinositide A, but with longer incubation periods the product is deacylated to isomeric forms of dimannophosphoinositides B and C.Brennan continued to work on these glycophosphoinositides after completing his postdoctoral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) and lipoarabinomannan (LAM)) were multiglycosylated extensions of Ballou''s phosphatidylinositol mannosides and are very important in the pathogenesis of tuberculosis and leprosy. More recent research has defined the biochemistry and genetics of synthesis of these molecules. Brennan is currently University Distinguished Professor in the Department of Microbiology, Immunology, and Pathology at Colorado State University. He served on the editorial board of the Journal of Biological Chemistry for several years and was also named Colorado State University Researcher of the Year in 1992.In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California, Berkeley, although he continued research and teaching for a few years. In recognition of his contributions to science, Ballou has received many awards and honors including election to the National Academy of Sciences (1975), the American Chemical Society''s Claude Hudson Award in Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the University of Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), a National Science Foundation Senior Fellowship (1961), and a University of California Berkeley Citation (1992). Ballou also served as an editorial board member for the Journal of Biological Chemistry.     

DNA Repair Mechanisms: the Work of Aziz Sancar

Action Mechanism of Escherichia coli DNA Photolyase. III. Photolysis of the Enzyme-Substrate Complex and the Absolute Action Spectrum(Sancar, G. B., Jorns, M. S., Payne, G., Fluke, D. J., Rupert, C. S., and Sancar, A. (1987) J. Biol. Chem. 262, 492–498)Reconstitution of the Human DNA Repair Excision Nuclease in the Highly Defined System(Mu, D., Park, C.-H., Matsunaga, T., Hsu, D. S., Reardon, J. T., and Sancar, A. (1995) J. Biol. Chem. 270, 2415–2418)Aziz Sancar was born in Savur, Turkey in 1946. Although both his parents were illiterate, they valued the importance of education and did their best to see that Sancar received a good one. They succeeded, and he excelled in many scientific subjects in high school. However, he also dreamed of playing on Turkey''s national soccer team, and this dream almost came true when, as a senior in high school, he was invited to attend tryouts to be a goalie on the national under-18 team. Ultimately he decided not to accept the invitation, later explaining, “upon serious consideration, I decided I wasn''t tall enough to be an outstanding goalie, and instead I concentrated on my studies” (1).Open in a separate windowAziz SancarAfter graduating in 1963, Sancar enrolled at Istanbul Medical School with the idea of becoming a doctor. However, after taking a biochemistry class during his 2nd year, he decided to become a research biochemist. Surprisingly, when he discussed his desire to pursue a Ph.D. with his biochemistry professor, he advised Sancar to practice medicine briefly before plunging into research, reasoning that anyone who spends the time getting a medical degree should at least practice for a couple years. So, Sancar spent 2 years as a rural physician near his hometown of Savur.In 1973, Sancar came to the United States to study with Claud Rupert in the molecular biology department of the University of Texas at Dallas. While in Turkey, Sancar had developed an interest in photoreactivation, the process by which DNA damaged by UV light is repaired by longer wavelength blue light. Rupert had identified photolyase, the enzyme that mediated the process by catalyzing the opening of the cyclobutane ring in pyrimidine dimers, and Sancar was eager to work him. The main topic of study in the Rupert laboratory in the early 1970s was the nature of photolyase''s chromophore. To that end, Sancar spent several years cloning and characterizing the gene for photolyase (2). After finally succeeding, he set out to purify the protein, but Rupert told him he had done enough research for his thesis and advised him to write his dissertation and graduate.After earning his Ph.D. in 1977, Sancar applied to three different laboratories hoping to continue studying DNA repair. All three laboratories rejected him. However, he learned that Dean Rupp at Yale University was interested in cloning repair genes, and although he didn''t have a postdoctoral position available, he was looking for a technician. Sancar accepted the job and joined the lab. Working with Rupp, Sancar identified and cloned several Escherichia coli repair genes, including the uvrA, uvrB, and uvrC genes involved in excision repair (3–5). He then purified the three uvr proteins and reconstituted the UVRABC nuclease, which he termed “excision nuclease” or “excinuclease” (6).In 1982, Sancar left Yale to become an associate professor of biochemistry at the University of North Carolina, Chapel Hill. There he resumed his work on photolyase and discovered that the enzyme contains two chromophores: FADH⁻ and a pterin (7–9). He also proposed a model for the reaction mechanism of photolyase repair, which is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here.At the time the Classic was published, there were two possible mechanisms for the repair reaction: the first involved energy transfer from a sensitizer to pyrimidine dimers, and the second involved electron transfer between the pyrimidine dimer and the photosensitizer. By determining the absolute action spectrum of the enzyme, Sancar and his colleagues were able to determine that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300–500 nm range, the photolyase chromophore donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. Eighteen years after publishing this Classic paper, Sancar was able to capture the excited flavin intermediate and observe the photolyase electron transfer, definitively proving his model (10).Sancar also continued studying other DNA repair pathways and soon turned his attention to excision repair in humans. The second JBC Classic is a result of Sancar''s studies on xeroderma pigmentosum, a hereditary disease caused by a defect in nucleotide excision repair as a result of mutations in one of several genes: XPA through XPG. In the paper, Sancar and his colleagues purified the components known to be required for the incision reaction and reconstituted the excision nuclease activity with these proteins. Using this system, they determined that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.Sancar is currently the Sarah Graham Kenan Professor of Biochemistry and Biophysics at the UNC School of Medicine. He has received many honors and awards in recognition of his contributions to science, including the Presidential Young Investigator Award from the National Science Foundation (1984) and the highest awards from the American Society for Photobiology (1990) and the Turkish Scientific Research Council (1995). Sancar was also the first Turkish-American member of the National Academy of Sciences (2005).     

Biotin-dependent Enzymes: the Work of Feodor Lynen

The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (+)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of the Reaction(Lane, M. D., Young, D. L., and Lynen, F. (1964)J. Biol. Chem.239, 2858–2864)Feodor Felix Konrad Lynen (1911–1979) was born in Munich, Germany. He was undecided about his career during his early education and even considered becoming a ski instructor. Ultimately, he enrolled in the Department of Chemistry at the University of Munich where he studied with Nobel laureate Heinrich Wieland and received his doctorate degree in 1937. Three months later, he married Wieland''s daughter, Eva.Open in a separate windowFeodor LynenAfter graduating, Lynen remained at Munich University as a postdoctoral fellow. He was appointed lecturer in 1942 and assistant professor in 1947. When World War II broke out, Lynen was exempt from military service because of a knee injury resulting from a ski accident in 1932. However, the war made it difficult to continue to do research in Munich, and Lynen moved his laboratory to the small village of Schondorf on the Ammersee. This was lucky because in 1945 Munich University''s Department of Chemistry was destroyed. Lynen continued his work at various laboratory facilities and eventually returned to the rebuilt Department of Chemistry in 1949.During the 1940s, Lynen began studying the biosynthesis of sterols and lipids. He eventually initiated a collaboration with Konrad Bloch, whose cholesterol research was featured in a previous Journal of Biological Chemistry (JBC) Classic (1). Working together, Bloch and Lynen were able to elucidate the steps in the biosynthesis of cholesterol. An especially significant finding made by Lynen was that acetyl coenzyme A (previously discovered by JBC Classic author Fritz Lipmann (2)) was essential for the first step of cholesterol biosynthesis. Lynen later determined the structure of acetyl-CoA. This work on cholesterol resulted in Bloch and Lynen being awarded the 1964 Nobel Prize in Physiology or Medicine.In 1953, Lynen was made full professor at the University of Munich. A year later, he was named director of the newly established Max Planck Institute for Cell Chemistry. He continued to work on fats but also turned his focus to biotin-dependent enzymes. In 1962, he was joined by JBC Classic author M. Daniel Lane (3), who had come to Munich to work with Lynen on a sabbatical leave. Lane was studying the biotin-dependent propionyl-CoA carboxylase and had previously determined that its biotin prosthetic group was linked to the enzyme through an amide linkage to a lysyl ϵ-amino group.Before leaving for Munich, Lane developed an apoenzyme system with which to investigate the mechanism by which biotin became attached to propionyl-CoA carboxylase. This system made use of Propionibacterium shermanii, which expressed huge amounts of methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had an absolute requirement for biotin in its growth medium and produced large amounts of the apotranscarboxylase when grown at very low levels of biotin.As reported in the JBC Classic reprinted here, Lane and Lynen were able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto the apoenzyme. Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, collaborated with them on these studies. In a second paper reprinted in the Lane Classic (4), Lane and Lynen showed that the synthetase catalyzed a two-step reaction. The first step involved the ATP-dependent formation of biotinyl-5′-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ϵ-amino group of the apotranscarboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1′-N position (5).In 1972, Lynen moved to the recently founded Max Planck Institute for Biochemistry. Between 1974 and 1976, he was acting director of the Institute while continuing to oversee a lab at the University of Munich. He remained at the Institute until his death in 1979.In addition to the Nobel Prize, Lynen received many honors and awards. These include the Neuberg Medal of the American Society of European Chemists and Pharmacists (1954), the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker (1955), the Carus Medal of the Deutsche Akademie der Naturforscher Leopoldina (1961), and the Otto Warburg Medal of the Gesellschaft für Physiologische Chemie (1963).     

Ligand Binding in Hemoglobin: the Work of Quentin H. Gibson

Oxygen Binding and Subunit Interaction of Hemoglobin in Relation to the Two-state Model(Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516–519)Ligand Recombination to the α and β Subunits of Human Hemoglobin(Olson, J. S., Rohlfs, R. J., and Gibson, Q. H. (1987) J. Biol. Chem. 262, 12930–12938)Quentin Howieson Gibson was born in Aberdeen, Scotland in 1918. He attended Queen''s University Belfast and received his M.B., Ch.B. degree in 1941, his M.D. in 1944, and his Ph.D. in 1946. After graduating, he became a lecturer in the Department of Physiology at the University of Sheffield and worked his way up to become Professor and Head of the Department of Biochemistry by 1955.Open in a separate windowQuentin H. GibsonIn 1963, Gibson came to the U.S. and joined the faculty of the Graduate School of Medicine at the University of Pennsylvania as a Professor of Physiology. He remained at Penn until 1965 when he became the Greater Philadelphia Professor of Biochemistry, Molecular and Cell Biology at Cornell University. In 1996, Gibson joined the Department of Biochemistry and Cell Biology at Rice University.Gibson is probably best known for his research on the structure and function of hemoglobin. The hemoglobin molecule consists of four globular protein subunits, each of which contains a heme group that can bind to one molecule of oxygen. The binding of oxygen to hemoglobin is cooperative, the first bound oxygen alters the shape of the molecule to increase the binding affinity of the additional subunits. Conversely, hemoglobin''s oxygen binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen.Gibson started his hemoglobin studies in graduate school, submitting a thesis titled “Methaemoglobin,” in which he studied the form of hemoglobin where the iron in the heme group is in the Fe³⁺ state rather than the Fe²⁺ state and is thus unable to carry oxygen. He followed this up with research on familial idiopathic methemoglobinemia, a hereditary hematological disease in which hemoglobin is unable to bind to oxygen, causing dyspnea and fatigue after physical exertion. He was able to identify the pathway involved in the reduction of methemoglobin (1), thereby describing the first hereditary disorder involving an enzyme deficiency. As a result, the disease was named “Gibson''s syndrome.” Since then, Gibson has made numerous additional contributions to the study of hemoglobin, some of which are detailed in the two Journal of Biological Chemistry (JBC) Classics reprinted here.In the first Classic, Gibson and Stuart J. Edelstein look at the oxygen binding and subunit interaction in hemoglobin. In 1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux proposed a model which stated that proteins that exhibit cooperativity can exist in only two conformational states, and the equilibrium between these two states is modified by binding of a ligand, oxygen in the case of hemoglobin (2). This became known as the “concerted” or the “MWC” model, for Monod, Wyman, and Changeux. (More information on Wyman''s research on protein chemistry and allosterism can be found in his JBC Classic (3).)By the mid-1980s, several groups had found evidence that challenged this model as it related to the mechanistic basis of ligand binding by hemoglobin. For example, Frederick C. Mills and Gary K. Ackers reported that the subunit interactions of hemoglobin decreased on binding of the fourth molecule of oxygen to hemoglobin (4). The effect, which they called “quaternary enhancement,” was incompatible with the two-state MWC allosteric model. In the first Classic, Gibson and Edelstein measured the free energy of binding of the fourth oxygen molecule and compared their result of −8.6 kcal/mol with Mills and Acker''s result of −9.3 kcal/mol. Gibson''s smaller value was consistent with other values found in the literature, and it also allowed reasonable representation of the equilibrium curve using the two-state model without invoking quaternary enhancement.In the second JBC Classic, Gibson looks at ligand binding in human hemoglobin. This paper was an extension of an analysis Gibson had done the previous year on ligand rebinding to sperm whale myoglobin (5). In the paper reprinted here, Gibson and his colleagues explored the rebinding of CO, O₂, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to the isolated α- and β-chains of hemoglobin as well as the intact molecule. From these experiments the researchers were able to determine the differences between the overall rate constants of the two hemoglobin subunits as well as the differences in binding of the various ligands.In recognition of his contributions to science, Gibson has earned many honors including memberships in the Royal Society of London, the National Academy of Sciences, and the American Association for the Advancement of Science. He served as an Associate Editor for the JBC from 1975 to 1994.     

Isolation Discovery of the Role of Dihydrotestosterone: the Work of Jean D. Wilson

The Conversion of Testosterone to 5α-Androstan-17β-ol-3-one by Rat Prostate in Vivo and in Vitro (Bruchovsky, N., and Wilson, J. D. (1968) J. Biol. Chem. 243, 2012–2021)The Intranuclear Binding of Testosterone and 5α-Androstan-17β-ol-3-one by Rat Prostate (Bruchovsky, N., and Wilson, J. D. (1968) J. Biol. Chem. 243, 5953–5960)Jean Donald Wilson was born in 1932 in a small town in the Texas Panhandle. He attended the University of Texas at Austin, and although he was a pre-med student, he decided to major in chemistry and minor in zoology. He graduated in 1951 and enrolled in medical school at the University of Texas Southwestern Medical Center at Dallas, where he spent a summer working with Donald W. Seldin on the effects of adrenal hormones on acid-base balance in the rat.Open in a separate windowJean D. WilsonAfter earning his M.D. in 1955, Wilson decided to stay in Dallas and did a residency in internal medicine at the Parkland Memorial Hospital. During this time, he spent 6 elective months working with Marvin D. Siperstein on the effects of diets high in saturated and unsaturated fats on the metabolism and excretion of cholesterol and bile acids in rats.When Wilson completed his residency in 1958, the physician''s draft was in effect, and he went to the National Institutes of Health to work as a clinical associate in the National Heart Institute. He spent most of his time doing clinical duties, but he also managed to spend part of each day working in the laboratory with Sidney Udenfriend, investigating the mechanism of ethanolamine biosynthesis.In 1960, Wilson returned to UT Southwestern as an instructor in the department of internal medicine, where he remains today as the Charles Cameron Sprague Distinguished Chair in Biomedical Science.Since starting his laboratory at UT Southwestern, Wilson''s research has focused on two areas. The first is cholesterol. Between 1960 and 1972, he developed methods for the quantification of cholesterol synthesis, absorption, degradation, and excretion in intact animals, with the aim of understanding the feedback control of cholesterol synthesis and turnover. He also demonstrated that plasma cholesterol is synthesized in the intestinal wall and liver, which led to the development of paradigms that defined the contributions of diet and endogenous synthesis to cholesterol turnover in humans and baboons.Wilson''s second research focus has been on hormone action, specifically the mechanisms by which steroid hormones influence protein turnover in the urogenital tract. A leading theory in the early 1960s was that steroid hormones regulate protein biosynthesis by controlling amino acid transport into cells (1). However, Wilson found that testosterone administration increased protein synthesis in the male urogenital tract prior to enhancement of amino acid transport, indicating that the increase in protein biosynthesis was secondary to an increase in RNA formation (2). He later showed that gonadal steroids are physically concentrated at the sites of mRNA synthesis in target tissues (3), but chromatin''s insolubility made it difficult to figure out to which macromolecule the hormones were attached.In 1966, Nicholas Bruchovsky joined Wilson''s lab as a postdoctoral fellow. His project was to determine whether a testosterone-binding protein could be isolated from prostatic nuclei. He injected animals with tritiated testosterone and used gel exclusion chromatography to show that radioactivity was bound to the nuclear components. Bruchovsky decided to confirm the identity of the bound hormone, but when he tried to isolate the radioactive nuclear material using thin layer chromatography, he was able to recover only a very small amount of it. By examining the chromatograms in discrete sections, Wilson and Bruchovsky discovered that the majority of radioactivity co-migrated with dihydrotestosterone, a potent metabolite of testosterone. Over the next several months, Wilson and Bruchovsky showed that the prostate contained enzymes that were very active in converting testosterone to dihydrotestosterone, and dihydrotestosterone to androstanediol, and managed to partially characterize testosterone 5α-reductase, the chromatin-associated nuclear enzyme that converts testosterone to dihydrotestosterone. They wrote up these results in a paper reprinted here as the first Journal of Biological Chemistry (JBC) Classic.This paper is the first to attach biological significance to the formation of dihydrotestosterone within target cells for testosterone. It became a Current Contents Citation Classic and was cited more than 640 times between 1968 and 1980.Wilson and Bruchovsky followed up this paper with the second JBC Classic in which they looked at the localization of dihydrotestosterone. They intravenously administered [l,2-³H]testosterone to rats and used gel filtration to examine the nuclear extracts. Their results confirmed that dihydrotestosterone was the predominant form of hormone bound to chromatin, proving that dihydrotestosterone is the active form of testosterone in peripheral tissues. Wilson went on to show that mutations in the steroid 5α-reductase gene cause a form of male pseudohermaphroditism in humans.Wilson is the recipient of several honors and awards, including the American Academy of Arts and Sciences Amory Prize (1977), the Society for Endocrinology Henry Dale Medal (1991), the Worcester Foundation for Experimental Biology Gregory Pincus Award (1992), the Endocrine Society Fred Conrad Koch Award (1993), and the Association of American Physicians Kober Medal (1999). He also is a member of the National Academy of Sciences, the Institute of Medicine, the American Philosophical Society, and the American Academy of Arts and Sciences, and he is a fellow of the Royal College of Physicians.¹     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.     

From discovering to understanding

Friedrich Miescher''s attempts to uncover the function of DNAIt might seem as though the role of DNA as the carrier of genetic information was not realized until the mid-1940s, when Oswald Avery (1877–1955) and colleagues demonstrated that DNA could transform bacteria (Avery et al, 1944). Although these experiments provided direct evidence for the function of DNA, the first ideas that it might have an important role in processes such as cell proliferation, fertilization and the transmission of heritable traits had already been put forward more than half a century earlier. Friedrich Miescher (1844–1895; Fig 1), the Swiss scientist who discovered DNA in 1869 (Miescher, 1869a), developed surprisingly insightful theories to explain its function and how biological molecules could encode information. Although his ideas were incorrect from today''s point of view, his work contains concepts that come tantalizingly close to our current understanding. But Miescher''s career also holds lessons beyond his scientific insights. It is the story of a brilliant scientist well on his way to making one of the most fundamental discoveries in the history of science, who ultimately fell short of his potential because he clung to established theories and failed to follow through with the interpretation of his findings in a new light.…a brilliant scientist well on his way to making one of the most fundamental discoveries in the history of science […] fell short of his potential because he clung to established theories…Open in a separate windowFigure 1Friedrich Miescher (1844–1895) and his wife, Maria Anna Rüsch. © Library of the University of Basel, Switzerland.It is a curious coincidence in the history of genetics that three of the most decisive discoveries in this field occurred within a decade: in 1859, Charles Darwin (1809–1882) published On the Origin of Species by Means of Natural Selection, in which he expounded the mechanism driving the evolution of species; seven years later, Gregor Mendel''s (1822–1884) paper describing the basic laws of inheritance appeared; and in early 1869, Miescher discovered DNA. Yet, although the magnitude of Darwin''s theory was realized almost immediately, and at least Mendel himself seems to have grasped the importance of his work, Miescher is often viewed as oblivious to the significance of his discovery. It would be another 75 years before Oswald Avery, Colin MacLeod (1909–1972) and Maclyn McCarthy (1911–2005) could convincingly show that DNA was the carrier of genetic information, and another decade before James Watson and Francis Crick (1916–2004) unravelled its structure (Watson & Crick, 1953), paving the way to our understanding of how DNA encodes information and how this is translated into proteins. But Miescher already had astonishing insights into the function of DNA.Between 1868 and 1869, Miescher worked at the University of Tübingen in Germany (Figs 2,​,3),3), where he tried to understand the chemical basis of life. A crucial difference in his approach compared with earlier attempts was that he worked with isolated cells—leukocytes that he obtained from pus—and later purified nuclei, rather than whole organs or tissues. The innovative protocols he developed allowed him to investigate the chemical composition of an isolated organelle (Dahm, 2005), which significantly reduced the complexity of his starting material and enabled him to analyse its constituents.Open in a separate windowFigure 2Contemporary view of the town of Tübingen at about the time when Miescher worked there. The medieval castle housing Hoppe-Seyler''s laboratory can be seen atop the hill at the right. © Stadtarchiv Tübingen, Germany.Open in a separate windowFigure 3The former kitchen of Tübingen castle, which formed part of Hoppe-Seyler''s laboratory. It was in this room that Miescher worked during his stay in Tübingen and where he discovered DNA. After his return to Basel, Miescher reminisced how this room with its shadowy, vaulted ceiling and its small, deep-set windows appeared to him like the laboratory of a medieval alchemist. Photograph taken by Paul Sinner, Tübingen, in 1879. © University Library Tübingen.In carefully designed experiments, Miescher discovered DNA—or “Nuclein” as he called it—and showed that it differed from the other classes of biological molecule known at that time (Miescher, 1871a). Most notably, nuclein''s elementary composition with its high phosphorous content convinced him that he had discovered a substance sui generis, that is, of its own kind; a conclusion subsequently confirmed by Miescher''s mentor in Tübingen, the eminent biochemist Felix Hoppe-Seyler (1825–1895; Hoppe-Seyler, 1871; Miescher, 1871a). After his initial analyses, Miescher was convinced that nuclein was an important molecule and suggested in his first publication that it would “merit to be considered equal to the proteins” (Miescher, 1871a).Moreover, Miescher recognized immediately that nuclein could be used to define the nucleus (Miescher, 1870). This was an important realization, as at the time the unequivocal identification of nuclei, and hence their study, was often difficult or even impossible to achieve because their morphology, subcellular localization and staining properties differed between tissues, cell types and states of the cells. Instead, Miescher proposed to base the characterization of nuclei on the presence of this molecule (Miescher, 1870, 1874). Moreover, he held that the nucleus should be defined by properties that are related to its physiological activity, which he believed to be closely linked to nuclein. Miescher had thus made a significant first step towards defining an organelle in terms of its function rather than its appearance.Importantly, his findings also showed that the nucleus is chemically distinct from the cytoplasm at a time when many scientists still assumed that there was nothing unique about this organelle. Miescher thus paved the way for the subsequent realization that cells are subdivided into compartments with distinct molecular composition and functions. On the basis of his observations that nuclein appeared able to separate itself from the “protoplasm” (cytoplasm), Miescher even went so far as to suggest the “possibility that [nuclein can be] distributed in the protoplasm, which could be the precursor for some of the de novo formations of nuclei” (Miescher, 1874). He seemed to anticipate that the nucleus re-forms around the chromosomes after cell division, but unfortunately did not elaborate on under which conditions this might occur. It is therefore impossible to know with certainty to which circumstances he was referring.Miescher thus paved the way for the subsequent realization that cells are subdivided into compartments with distinct molecular composition and functionsIn this context, it is interesting to note that in 1872, Edmund Russow (1841–1897) observed that chromosomes appeared to dissolve in basic solutions. Intriguingly, Miescher had also found that he could precipitate nuclein by using acids and then return it to solution by increasing the pH (Miescher, 1871a). At the time, however, he did not make the link between nuclein and chromatin. This happened around a decade later, in 1881, when Eduard Zacharias (1852–1911) studied the nature of chromosomes by using some of the same methods Miescher had used when characterizing nuclein. Zacharias found that chromosomes, such as nuclein, were resistant to digestion by pepsin solutions and that the chromatin disappeared when he extracted the pepsin-treated cells with dilute alkaline solutions. This led Walther Flemming (1843–1905) to speculate in 1882 that nuclein and chromatin are identical (Mayr, 1982).Alas, Miescher was not convinced. His reluctance to accept these developments was at least partly based on a profound scepticism towards the methods—and hence results—of cytologists and histologists, which, according to Miescher, lacked the precision of chemical approaches as he applied them. The fact that DNA was crucially linked to the function of the nucleus was, however, firmly established in Miescher''s mind and in the following years he tried to obtain additional evidence. He later wrote: “Above all, using a range of suitable plant and animal specimens, I want to prove that Nuclein really specifically belongs to the life of the nucleus” (Miescher, 1876).Although the acidic nature of DNA, its large molecular weight, elementary composition and presence in the nucleus are some of its central properties—all first determined by Miescher—they reveal nothing about its function. Having convinced himself that he had discovered a new type of molecule, Miescher rapidly set out to understand its role in different biological contexts. As a first step, he determined that nuclein occurs in a variety of cell types. Unfortunately, he did not elaborate on the types of tissue or the species his samples were derived from. The only hints as to the specimens he worked with come from letters he wrote to his uncle, the Swiss anatomist Wilhelm His (1831–1904), and his parents; his father, Friedrich Miescher-His (1811–1887), was professor of anatomy in Miescher''s native Basel. In his correspondence, Miescher mentioned other cell types that he had studied for the presence of nuclein, including liver, kidney, yeast cells, erythrocytes and chicken eggs, and hinted at having found nuclein in these as well (Miescher, 1869b; His, 1897). Moreover, Miescher had also planned to look for nuclein in plants, especially in their spores (Miescher, 1869c). This is an intriguing choice given his later fascination with vertebrate germ cells and his speculation on the processes of fertilization and heredity (Miescher, 1871b, 1874).Another clue to the tissues and cell types that Miescher might have examined comes from two papers published by Hoppe-Seyler, who wanted to confirm his student''s results, which he initially viewed with scepticism, before their publication. In the first, another of Hoppe-Seyler''s students, Pal Plósz, reported that nuclein is present in the nucleated erythrocytes of snakes and birds but not in the anuclear erythrocytes of cows (Plósz, 1871). In the second paper, Hoppe-Seyler himself confirmed Miescher''s findings and reported that he had detected nuclein in yeast cells (Hoppe-Seyler, 1871).In an addendum to his 1871 paper, published posthumously, Miescher stated that the apparently ubiquitous presence of nuclein meant that “a new factor has been found for the life of the most basic as well as for the most advanced organisms,” thus opening up a wide range of questions for physiology in general (Miescher, 1870). To argue that Miescher understood that DNA was an essential component of all forms of life is probably an over-interpretation of his words. His statement does, however, clearly show that he believed DNA to be an important factor in the life of a wide range of species.In addition, Miescher looked at tissues under different physiological conditions. He quickly noticed that both nuclein and nuclei were significantly more abundant in proliferating tissues; for instance, he noted that in plants, large amounts of phosphorous are found predominantly in regions of growth and that these parts show the highest densities of nuclei and actively proliferating cells (Miescher, 1871a). Miescher had thus taken the first step towards linking the presence of phosphorous—that is, DNA in this context—to cell proliferation. Some years later, while examining changes in the bodies of salmon as they migrate upstream to their spawning grounds, he noticed that he could, with minimal effort, purify large amounts of pure nuclein from the testes, as they were at the height of cell proliferation in preparation for mating (Miescher, 1874). This provided additional evidence for a link between proliferation and the presence of a high concentration of nuclein.Miescher''s most insightful comments on this issue, however, date from his time in Hoppe-Seyler''s laboratory in Tübingen. He was convinced that histochemical analyses would lead to a much better understanding of certain pathological states than would microscopic studies. He also believed that physiological processes, which at the time were seen as similar, might turn out to be very different if the chemistry were better understood. As early as 1869, the year in which he discovered nuclein, he wrote in a letter to His: “Based on the relative amounts of nuclear substances [DNA], proteins and secondary degradation products, it would be possible to assess the physiological significance of changes with greater accuracy than is feasible now” (Miescher, 1869c).Importantly, Miescher proposed three exemplary processes that might benefit from such analyses: “nutritive progression”, characterized by an increase in the cytoplasmic proteins and the enlargement of the cell; “generative progression”, defined as an increase in “nuclear substances” (nuclein) and as a preliminary phase of cell division in proliferating cells and possibly in tumours; and “regression”, an accumulation of lipids and degenerative products (Miescher, 1869c).When we consider the first two categories, Miescher seems to have understood that an increase in DNA was not only associated with, but also a prerequisite for cell proliferation. Subsequently, cells that are no longer proliferating would increase in size through the synthesis of proteins and hence cytoplasm. Crucially, he believed that chemical analyses of such different states would enable him to obtain a more fundamental insight into the causes underlying these processes. These are astonishingly prescient insights. Sadly, Miescher never followed up on these ideas and, apart from the thoughts expressed in his letter, never published on the topic.…Miescher seems to have understood that an increase in DNA was not only associated with, but also a prerequisite for cell proliferationIt is likely, however, that he had preliminary data supporting these views. Miescher was generally careful to base statements on facts rather than speculation. But, being a perfectionist who published only after extensive verification of his results, he presumably never pursued these studies to such a satisfactory point. It is possible his plans were cut short by leaving Hoppe-Seyler''s laboratory to receive additional training under the supervision of Carl Ludwig (1816–1895) in Leipzig. While there, Miescher turned his attention to matters entirely unrelated to DNA and only resumed his work on nuclein after returning to his native Basel in 1871.Crucially for these subsequent studies of nuclein, Miescher made an important choice: he turned to sperm as his main source of DNA. When analysing the sperm from different species, he noted that the spermatozoa, especially of salmon, have comparatively small tails and thus consist mainly of a nucleus (Miescher, 1874). He immediately grasped that this would greatly facilitate his efforts to isolate DNA at much higher purity (Fig 4). Yet, Miescher also saw beyond the possibility of obtaining pure nuclein from salmon sperm. He realized it also indicated that the nucleus and the nuclein therein might play a crucial role in fertilization and the transmission of heritable traits. In a letter to his colleague Rudolf Boehm (1844–1926) in Würzburg, Miescher wrote: “Ultimately, I expect insights of a more fundamental importance than just for the physiology of sperm” (Miescher, 1871c). It was the beginning of a fascination with the phenomena of fertilization and heredity that would occupy Miescher to the end of his days.Open in a separate windowFigure 4A glass vial containing DNA purified by Friedrich Miescher from salmon sperm. © Alfons Renz, University of Tübingen, Germany.Miescher had entered this field at a critical time. By the middle of the nineteenth century, the old view that cells arise through spontaneous generation had been challenged. Instead, it was widely recognized that cells always arise from other cells (Mayr, 1982). In particular, the development and function of spermatozoa and oocytes, which in the mid-1800s had been shown to be cells, were seen in a new light. Moreover, in 1866, three years before Miescher discovered DNA, Ernst Haeckel (1834–1919) had postulated that the nucleus contained the factors that transmit heritable traits. This proposition from one of the most influential scientists of the time brought the nucleus to the centre of attention for many biologists. Having discovered nuclein as a distinctive molecule present exclusively in this organelle, Miescher realized that he was in an excellent position to make a contribution to this field. Thus, he set about trying to better characterize nuclein with the aim of correlating its chemical properties with the morphology and function of cells, especially of sperm cells.His analyses of the chemical composition of the heads of salmon spermatozoa led Miescher to identify two principal components: in addition to the acidic nuclein, he found an alkaline protein for which he coined the term ‘protamin''; the name is still in use today; protamines are small proteins that replace histones during spermatogenesis. He further determined that these two molecules occur in a “salt-like, not an ether-like [that is, covalent] association” (Miescher, 1874). Following his meticulous analyses of the chemical composition of sperm, he concluded that, “aside from the mentioned substances [protamin and nuclein] nothing is present in significant quantity. As this is crucial for the theory of fertilization, I carry this business out as quantitatively as possible right from the beginning” (Miescher, 1872a). His analyses showed him that the DNA and protamines in sperm occur at constant ratios; a fact that Miescher considered “is certainly of special importance,” without, however, elaborating on what might be this importance. Today, of course, we know that proteins, such as histones and protamines, bind to DNA in defined stoichiometric ratios.Miescher went on to analyse the spermatozoa of carp, frogs (Rana esculenta) and bulls, in which he confirmed the presence of large amounts of nuclein (Miescher, 1874). Importantly, he could show that nuclein is present in only the heads of sperm—the tails being composed largely of lipids and proteins—and that within the head, the nuclein is located in the nucleus (Miescher, 1874; Schmiedeberg & Miescher, 1896). With this discovery, Miescher had not only demonstrated that DNA is a constant component of spermatozoa, but also directed his attention to the sperm heads. On the basis of the observations of other investigators, such as those of Albert von Kölliker (1817–1905) concerning the morphology of spermatozoa in some myriapods and arachnids, Miescher knew that the spermatozoa of some species are aflagellate, that is, lack a tail. This confirmed that the sperm head, and thus the nucleus, was the crucial component. But, the question remained: what in the sperm cells mediated fertilization and the transmission of hereditary traits from one generation to the next?On the basis of his chemical analyses of sperm, Miescher speculated on the existence of molecules that have a crucial part in these processes. In a letter to Boehm, Miescher wrote: “If chemicals do play a role in procreation at all, then the decisive factor is now a known substance” (Miescher, 1872b). But Miescher was unsure as to what might be this substance. He did, however, strongly suspect the combination of nuclein and protamin was the key and that the oocyte might lack a crucial component to be able to develop: “If now the decisive difference between the oocyte and an ordinary cell would be that from the roster of factors, which account for an active arrangement, an element has been removed? For otherwise all proper cellular substances are present in the egg,” he later wrote (Miescher, 1872b).Owing to his inability to detect protamin in the oocyte, Miescher initially favoured this molecule as responsible for fertilization. Later, however, when he failed to detect protamin in the sperm of other species, such as bulls, he changed his mind: “The Nuclein by contrast has proved to be constant [that is, present in the sperm cells of all species Miescher analysed] so far; to it and its associations I will direct my interest from now on” (Miescher, 1872b). Unfortunately, however, although he came tantalizingly close, he never made a clear link between nuclein and heredity.The final section of his 1874 paper on the occurrence and properties of nuclein in the spermatozoa of different vertebrate species is of particular interest because Miescher tried to correlate his chemical findings about nuclein with the physiological role of spermatozoa. He had realized that spermatozoa represented an ideal model system to study the role of DNA because, as he would later put it, “[f]or the actual chemical–biological problems, the great advantage of sperm [cells] is that everything is reduced to the really active substances and that they are caught just at the moment when they exert their greatest physiological function” (Miescher, 1893a). He appreciated that his data were still incomplete, yet wanted to make a first attempt to pull his results together and integrate them into a broader picture to explain fertilization.At the time, Wilhelm Kühne (1837–1900), among others, was putting forward the idea that spermatozoa are the carriers of specific substances that, through their chemical properties, achieve fertilization (Kühne, 1868). Miescher considered his results of the chemical composition of spermatozoa in this context. While critically considering the possibility of a chemical substance explaining fertilization, he stated that: “if we were to assume at all that a single substance, as an enzyme or in any other way, for instance as a chemical impulse, could be the specific cause of fertilization, we would without a doubt first and foremost have to consider Nuclein. Nuclein-bodies were consistently found to be the main components [of spermatozoa]” (Miescher, 1874).With hindsight, these statements seem to suggest that Miescher had identified nuclein as the molecule that mediates fertilization—a crucial assumption to follow up on its role in heredity. Unfortunately, however, Miescher himself was far from convinced that a molecule (or molecules) was responsible for this. There are several reasons for his reluctance, although the influence of his uncle was presumably a crucial factor as it was he who had been instrumental in fostering the young Miescher''s interest in biochemistry and remained a strong influence throughout his life. Indeed, when Miescher came tantalizingly close to uncovering the function of DNA, His''s views proved counterproductive, probably preventing him from interpreting his findings in the context of new results from other scientists at the time. Miescher thus failed to take his studies of nuclein and its function in fertilization and heredity to the next level, which might well have resulted in recognizing DNA as the central molecule in both processes.One specific aspect that diverted Miescher from contemplating the role of nuclein in fertilization was a previous study in which he had erroneously identified the yolk platelets in chicken oocytes as a large number of nuclein-containing granules (Miescher, 1871b). This led him to conclude that the comparatively minimal quantitative contribution of DNA from a spermatozoon to an oocyte, which already contained so much more of the substance, could not have a significant impact on the latter''s physiology. He therefore concluded that, “not in a specific substance can the mystery of fertilization be concealed. […] Not a part, but the whole must act through the cooperation of all its parts” (Miescher, 1874).It is all the more unfortunate that Miescher had identified the yolk platelets in oocytes as nuclein-containing cells because he had realized that the presumed nuclein in these granules differed from the nuclein (that is, DNA) he had isolated previously from other sources, notably by its much higher phosphorous content. But influenced by His''s strong view that these structures were genuine cells, Miescher saw his results in this light. Only several years later, based on results from his contemporaries Flemming and Eduard A. Strasburger (1844–1912) on the morphological properties of nuclei and their behaviour during cell divisions, and Albrecht Kossel''s (1853–1927) discoveries about the composition of DNA (Portugal & Cohen, 1977), did Miescher revise his initial assumption that chicken oocytes contain a large number of nuclein-containing granules. Instead, he finally conceded that the molecules comprising these granules were different from nuclein (Miescher, 1890).Another factor that prevented Miescher from concluding that nuclein was the basis for the transmission of hereditary traits was that he could not conceive of how a single substance might explain the multitude of heritable traits. How, he wondered, could a specific molecule be responsible for the differences between species, races and individuals? Although he granted that “differences in the chemical constitution of these molecules [different types of nuclein] will occur, but only to a limited extent” (Miescher, 1874).And thus, instead of looking to molecules, he—like his uncle His––favoured the idea that the physical movement of the sperm cells or an activation of the oocyte, which he likened to the stimulation of a muscle by neuronal impulses, was responsible for the process of fertilization: “Like the muscle during the activation of its nerve, the oocyte will, when it receives appropriate impulses, become a chemically and physically very different entity” (Miescher, 1874). For nuclein itself, Miescher considered that it might be a source material for other molecules, such as lecithin––one of the few other molecules with a high phosphorous content known at the time (Miescher, 1870, 1871a, 1874). Miescher clearly preferred the idea of nuclein as a repository for material for the cell—mainly phosphorous—rather than as a molecule with a role in encoding information to synthesize such materials. This idea of large molecules being source material for smaller ones was common at the time and was also contemplated for proteins (Miescher, 1870).The entire section of Miescher''s 1874 paper in which he discusses the physiological role of nuclein reads as though he was deliberately trying to assemble evidence against nuclein being the key molecule in fertilization and heredity. This disparaging approach towards the molecule that he himself had discovered might also be explained, at least to some extent, by his pronounced tendency to view his results so critically; tellingly, he published only about 15 papers and lectures in a career spanning nearly three decades.The modern understanding that fertilization is achieved by the fusion of two germ cells only became established in the final quarter of the nineteenth century. Before that time, the almost ubiquitous view was that the sperm cell, through mere contact with the egg, in some way stimulated the oocyte to develop—the physicalist viewpoint. His was a key advocate of this view and firmly rejected the idea that a specific substance might mediate heredity. We can only speculate as to how Miescher would have interpreted his results had he worked in a different intellectual environment at the time, or had he been more independent in the interpretation of his results.We can only speculate as to how Miescher would have interpreted his results had he worked in a different intellectual environment at the time…Miescher''s refusal to accept nuclein as the key to fertilization and heredity is particularly tragic in view of several studies that appeared in the mid-1870s, focusing the attention of scientists on the nuclei. Leopold Auerbach (1828–1897) demonstrated that fertilized eggs contain two nuclei that move towards each other and fuse before the subsequent development of the embryo (Auerbach, 1874). This observation strongly suggested an important role for the nuclei in fertilization. In a subsequent study, Oskar Hertwig (1849–1922) confirmed that the two nuclei—one from the sperm cell and one from the oocyte—fuse before embryogenesis begins. Furthermore, he observed that all nuclei in the embryo derive from this initial nucleus in the zygote (Hertwig, 1876). With this he had established that a single sperm fertilizes the oocyte and that there is a continuous lineage of nuclei from the zygote throughout development. In doing so, he delivered the death blow to the physicalist view of fertilization.By the mid-1880s, Hertwig and Kölliker had already postulated that the crucial component of the nucleus that mediated inheritance was nuclein—an idea that was subsequently accepted by several scientists. Sadly, Miescher remained doubtful until his death in 1895 and thus failed to appreciate the true importance of his discovery. This might have been an overreaction to the claims by others that sperm heads are formed from a homogeneous substance; Miescher had clearly shown that they also contain other molecules, such as proteins. Moreover, Miescher''s erroneous assumption that nuclein occurred only in the outer shell of the sperm head resulted in his failure to realize that stains for chromatin, which stain the centres of the heads, actually label the region where there is nuclein; although he later realized that virtually the entire sperm head is composed of nuclein and associated protein (Miescher, 1892a; Schmiedeberg & Miescher, 1896).Unfortunately, not only Miescher, but the entire scientific community would soon lose faith in DNA as the molecule mediating heredity. Miescher''s work had established DNA as a crucial component of all cells and inspired others to begin exploring its role in heredity, but with the emergence of the tetranucleotide hypothesis at the beginning of the twentieth century, DNA fell from favour and was replaced by proteins as the prime candidates for this function. The tetranucleotide hypothesis—which assumed that DNA was composed of identical subunits, each containing all four bases—prevailed until the late 1940s when Edwin Chargaff (1905–2002) discovered that the different bases in DNA were not present in equimolar amounts (Chargaff et al, 1949, 1951).Unfortunately, not only Miescher, but the entire scientific community would soon lose faith in DNA as the molecule mediating heredityJust a few years before, in 1944, experiments by Avery and colleagues had demonstrated that DNA was sufficient to transform bacteria (Avery et al, 1944). Then in 1952, Al Hershey (1908–1997) and Martha Chase (1927–2003) confirmed these findings by observing that viral DNA—but no protein—enters the bacteria during infection with the T2 bacteriophage and, that this DNA is also present in new viruses produced by infected bacteria (Hershey & Chase, 1952). Finally, in 1953, X-ray images of DNA allowed Watson and Crick to deduce its structure (Watson & Crick, 1953) and thus enable us to understand how DNA works. Importantly, these experiments were made possible by advances in bacteriology and virology, as well as the development of new techniques, such as the radioactive labelling of proteins and nucleic acids, and X-ray crystallography—resources that were beyond the reach of Miescher and his contemporaries.In later years (Fig 5), Miescher''s attention shifted progressively from the role of nuclein in fertilization and heredity to physiological questions, such as those concerning the metabolic changes in the bodies of salmon as they produce massive amounts of germ cells at the expense of muscle tissue. Although he made important and seminal contributions to different areas of physiology, he increasingly neglected to explore his most promising line of research, the function of DNA. Only towards the end of his life did he return to this question and begin to reconsider the issue in a new light, but he achieved no further breakthroughs.Open in a separate windowFigure 5Friedrich Miescher in his later years when he was Professor of Physiology at the University of Basel. In this capacity he also founded the Vesalianum, the University''s Institute for Anatomy and Physiology, which was inaugurated in 1885. This photograph is the frontispiece on the inside cover of a collection of Miescher''s publications and some of his letters, edited and published by his uncle Wilhelm His and colleagues after Miescher''s death. Underneath the picture is Miescher''s signature. © Ralf Dahm.One area, however, where he did propose intriguing hypotheses—although without experimental data to support them—was the molecular underpinnings of heredity. Inspired by Darwin''s work on fertilization in plants, Miescher postulated, for instance, how information might be encoded in biological molecules. He stated that, “the key to sexuality for me lies in stereochemistry,” and expounded his belief that the gemmules of Darwin''s theory of pangenesis were likely to be “numerous asymmetric carbon atoms [present in] organic substances” (Miescher, 1892b), and that sexual reproduction might function to correct mistakes in their “stereometric architecture”. As such, Miescher proposed that hereditary information might be encoded in macromolecules and how mistakes could be corrected, which sounds uncannily as though he had predicted what is now known as the complementation of haploid deficiencies by wild-type alleles. It is particularly tempting to assume that Miescher might have thought this was the case, as Mendel had published his laws of inheritance of recessive characteristics more than 25 years earlier. However, there is no reference to Mendel''s work in the papers, talks or letters that Miescher has left to us.Miescher proposed that hereditary information might be encoded in macromolecules and how mistakes could be corrected…What we do know is that Miescher set out his view of how hereditary information might be stored in macromolecules: “In the enormous protein molecules […] the many asymmetric carbon atoms allow such a colossal number of stereoisomers that all the abundance and diversity of the transmission of hereditary [traits] may find its expression in them, as the words and terms of all languages do in the 24–30 letters of the alphabet. It is therefore completely superfluous to see the sperm cell or oocyte as a repository of countless chemical substances, each of which should be the carrier of a special hereditary trait (de Vries Pangenesis). The protoplasm and the nucleus, that my studies have shown, do not consist of countless chemical substances, but of very few chemical individuals, which, however, perhaps have a very complex chemical structure” (Miescher, 1892b).This is a remarkable passage in Miescher''s writings. The second half of the nineteenth century saw intense speculation about how heritable characteristics are transmitted between the generations. The consensus view assumed the involvement of tiny particles, which were thought to both shape embryonic development and mediate inheritance (Mayr, 1982). Miescher contradicted this view. Instead of a multitude of individual particles, each of which might be responsible for a specific trait (or traits), his results had shown that, for instance, the heads of sperm cells are composed of only very few compounds, chiefly DNA and associated proteins.He elaborated further on his theory of how hereditary information might be stored in large molecules: “Continuity does not only lie in the form, it also lies deeper than the chemical molecule. It lies in the constituent groups of atoms. In this sense I am an adherent of a chemical model of inheritance à outrance [to the utmost]” (Miescher, 1893b). With this statement Miescher firmly rejects any idea of preformation or some morphological continuity transmitted through the germ cells. Instead, he clearly seems to foresee what would only become known much later: the basis of heredity was to be found in the chemical composition of molecules.To explain how this could be achieved, he proposed a model of how information could be encoded in a macromolecule: “If, as is easily possible, a protein molecule comprises 40 asymmetric carbon atoms, there will be 2⁴⁰, that is, approximately a trillion isomerisms [sic]. And this is only one of the possible types of isomerism [not considering other atoms, such as nitrogen]. To achieve the incalculable diversity demanded by the theory of heredity, my theory is better suited than any other. All manner of transitions are conceivable, from the imperceptible to the largest differences” (Miescher, 1893b).Miescher''s ideas about how heritable characteristics might be transmitted and encoded encapsulate several important concepts that have since been proven to be correct. First, he believed that sexual reproduction served to correct mistakes, or mutations as we call them today. Second, he postulated that the transmission of heritable traits occurs through one or a few macromolecules with complex chemical compositions that encode the information, rather than by numerous individual molecules each encoding single traits, as was thought at the time. Third, he foresaw that information is encoded in these molecules through a simple code that results in a staggeringly large number of possible heritable traits and thus explain the diversity of species and individuals observed.Miescher''s ideas about how heritable characteristics might be transmitted and encoded encapsulate several important concepts that have since been proven to be correctIt is a step too far to suggest that Miescher understood what DNA or other macromolecules do, or how hereditary information is stored. He simply could not have done, given the context of his time. His findings and hypotheses that today fit nicely together and often seem to anticipate our modern understanding probably appeared rather disjointed to Miescher and his contemporaries. In his day, too many facts were still in doubt and too many links tenuous. There is always a danger of over-interpreting speculations and hypotheses made a long time ago in today''s light. However, although Miescher himself misinterpreted some of his findings, large parts of his conclusions came astonishingly close to what we now know to be true. Moreover, his work influenced others to pursue their own investigations into DNA and its function (Dahm, 2008). Although DNA research fell out of fashion for several decades after the end of the nineteenth century, the information gleaned by Miescher and his contemporaries formed the foundation for the decisive experiments carried out in the middle of the twentieth century, which unambiguously established the function of DNA.As such, perhaps the most tragic aspect of Miescher''s career was that for most of his life he firmly believed in the physicalist theories of fertilization, as propounded by His and Ludwig among others, and his reluctance to combine the results from his rigorous chemical analyses with the ‘softer'' data generated by cytologists and histologists. Had he made the link between nuclein and chromosomes and accepted its key role in fertilization and heredity, he might have realized that the molecule he had discovered was the key to some of the greatest mysteries of life. As it was, he died with a feeling of a promising career unfulfilled (His, 1897), when, in actual fact, his contributions were to outshine those of most of his contemporaries.…he died with a feeling of a promising career unfulfilled […] when, in actual fact, his contributions were to outshine those of most of his contemporariesIt is tantalizing to speculate the path that Miescher''s investigations—and biology as a whole—might have taken under slightly different circumstances. What would have happened had he followed up on his preliminary results about the role of DNA in different physiological conditions, such as cell proliferation? How would his theories about fertilization and heredity have changed had he not been misled by the mistaken identification of what appeared to him to be a multitude of small nuclei in the oocyte? Or how would he have interpreted his findings concerning nuclein had he not been influenced by the views of his uncle, but also those of the wider scientific establishment?There is a more general lesson in the life and work of Friedrich Miescher that goes beyond his immediate successes and failures. His story is that of a brilliant researcher who developed innovative experimental approaches, chose the right biological systems to address his questions and made ground-breaking discoveries, and who was nonetheless constrained by his intellectual environment and thus prevented from interpreting his findings objectively. It therefore fell to others, who saw his work from a new point of view, to make the crucial inferences and thus establish the function of DNA.? Open in a separate windowRalf Dahm