Squalene synthase-deficient mutant of Chinese hamster ovary cells.

Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

Multienzyme Complexes and Hydrogen Transfer: the Work of Perry Frey

Quaternary Structure of Pyruvate Dehydrogenase Complex from Esherichia coli(Yang, H. C., Hainfeld, J. F., Wall, J. S., and Frey, P. A. (1985) J. Biol. Chem. 260, 16049–16051)Lysine 2,3-Aminomutase. Support for a Mechanism of Hydrogen Transfer Involving S-Adenosylmethionine(Baraniak, J., Moss, M. L., and Frey, P. A. (1989) J. Biol. Chem. 264, 1357–1360)Perry A. Frey was born in 1935 in Plain City, Ohio, a small town about 18 miles northwest of Columbus. Prior to attending college he served in the military for 2 years. He then enrolled at Ohio State University where he received his B.S. in chemistry in 1959. After graduating, Frey worked for the United States Public Health Service as an analytical chemist, studying the properties of saxitoxin, a paralytic shellfish poison. He also attended night classes in chemistry at the University of Cincinnati.Open in a separate windowPerry FreyIn 1964, based on the recommendation of a mentor at the Public Health Service, Frey decided to go to graduate school at the University of Michigan to work with Robert H. Abeles. After a short time, Abeles moved his laboratory to Brandeis University and Frey went with him. Frey spent 3 years studying catalysis by cobalamin-dependent enzymes and earned his Ph.D in biochemistry in 1968. He then began a postdoctoral fellowship at Harvard University with enzymologist Frank H. Westheimer.In 1969, Frey accepted an offer to join the chemistry department at Ohio State University. He remained there for several years, rising through the ranks to eventually become Professor of Biochemistry and Academic Vice Chair of Chemistry. During his time at Ohio State, Frey started investigating the mechanism of enzyme and coenzyme action in several molecules, including UDP-galactose 4-epimerase, pyruvate dehydrogenase, galactose-1-phosphate uridylyltransferase, UDP-glucose pyrophosphorylase, and adenylate kinase.In 1981, Frey left Ohio State to join the faculty of the Institute for Enzyme Research at the University of Wisconsin, Madison. There he continued to study enzyme mechanisms but also expanded his research to include the structure and function of multienzyme complexes. In the first Journal of Biological Chemistry (JBC) Classic reprinted here, Frey reports the results of his quaternary structural analysis of the pyruvate dehydrogenase complex from Escherichia coli. Using scanning transmission electron microscopy and radial mass analysis, Frey and his colleagues were able to confirm a model in which six dihydrolipoyl dehydrogenase (E₃) dimers are integrated into the six faces of a cubic dihydrolipoyl transacetylase (E₂) core and 12 pyruvate dehydrogenase (E₁) dimers are associated along the 12 edges of the core enzyme.Frey also began to work on lysine 2,3-aminomutase, the enzyme that catalyzed the conversion of l-lysine to l-β-lysine. The reaction involved the interchange of the 2-amino group of lysine with a hydrogen at carbon 3 to form β-lysine and was analogous to adenosylcobalamin-dependent rearrangements in which hydrogen transfer is mediated by the adenosyl moiety of the coenzyme. However, lysine 2,3-aminomutase did not appear to contain adenosylcobalamin, and it wasn''t activated by the coenzyme. To explain this phenomenon, Frey suggested that S-adenosylmethionine was involved in the hydrogen transfer reaction and proposed a mechanism in which the adenosyl-C-5′ moiety of S-adenosylmethionine functioned in the same way as the adenosyl group of adenosylcobalamin in facilitating hydrogen transfer and generating an intermediate free radical that could undergo the amino group migration (1).In the second JBC Classic reprinted here, Frey and his colleagues further tested his proposed mechanism and, by carrying out the lysine 2,3-aminomutase reaction with S-[5′-³H]adenosylmethionine, showed that both hydrogens at adenosyl-C-5′ participate in the hydrogen transfer process. Mass spectral analysis of the β-lysine for monodeutero and dideutero species also demonstrated that the hydrogen transfer is both intramolecular and intermolecular. The results of this paper confirmed that the activation of lysine 2,3-aminomutase involved a transformation of S-adenosylmethionine into a form that promotes the generation of a 5′-adenosyl free radical which abstracts hydrogen from lysine to form 5′-deoxyadenosine as an intermediate.Frey retired in 2008 and is currently an emeritus professor in the biochemistry department at the University of Wisconsin, Madison. In recognition of his contributions to science, he has received many honors and awards including the Alexander von Humboldt Senior Scientist Award (1995), the American Chemical Society Division of Biological Chemistry''s Repligen Award (2000), and the Hilldale Award (2007). Frey is a fellow of the American Association for the Advancement of Science (2003) and the American Academy of Arts and Sciences (2003) and was elected to the National Academy of Sciences (1998). He also served on the editorial board for the Journal Biological Chemistry from 1983 to 1988.     

A topological analysis of subunit alpha from Escherichia coli F1F0-ATP synthase predicts eight transmembrane segments

The membrane topology of subunit alpha from the Escherichia coli F1F0-ATP synthase was studied using a gene fusion technique. Fusion proteins linking different amino-terminal fragments of the alpha subunit with an enzymatically active fragment of alkaline phosphatase were constructed by both random transposition of TnphoA and site-directed mutagenesis. Those proteins with high levels of alkaline phosphatase activity are predicted to define periplasmic domains of alpha, and this was confirmed by testing for cell growth in minimal medium supplemented with polyphosphate (P greater than 75) as the sole source of phosphate. The enzymatic activity of some fusion proteins was shown to be sensitive to glucose present in the growth medium. Results from subcellular fractionation experiments suggest that these fusion proteins may be inactive even though they have a periplasmic alkaline phosphatase. The enzymatic activity appears dependent upon proteolytic release of the alkaline phosphatase moiety from its alpha subunit membrane anchor and suggests the target of glucose repression may be a protease present in the periplasm. For the topological analysis of the alpha subunit, a total of 28 unique fusion proteins were studied and the results were consistent with a model of alpha containing eight transmembrane segments, including periplasmic amino and carboxyl termini. Surprisingly, separate periplasmic domains were identified near amino acids 200, 233, and 270. These results suggest the flanking membrane spans are only 10-15 amino acids in length and not able to span a standard 30 A bilayer in an alpha-helical conformation. These short spans may have interesting mechanistic implications for the function of F0, because they contain several amino acids which appear critical for proton translocation. Finally, a fusion of alkaline phosphatase at amino acid 271, the carboxyl-terminal residue, but not at amino acid 260, was able to complement the strain RH305 (uncB-) for growth on succinate and suggests the last 11 amino acids of the alpha subunit are critical to the function of F1F0-ATP synthase.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Similar Ca(2+)-signaling properties in keratinocytes and in COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase SPCA1

Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.