A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung

Mouse Ehrlich ascites tumor cells incubated with the creatine analog, N-ethylguanidinoacetate (N-amidino-N-ethylglycine), accumulated up to 8 μmol/g packed cells of the creatine-P analog, N-ethylguanidinoacetate-P. Evaluation of N-ethylguanidinoacetate-P as a synthetic phosphagen under in vivo conditions was performed with Ehrlich cells loaded with equimolar amounts of a common reference phosphagen, cyclocreatine-P (1-carboxymethyl-2-imino-3-phosphonoimidazolidine) plus either N-ethylguanidinoacetate-P or creatine-P. It was concluded that N-ethylguanidinoacetate-P has a Gibbs free energy of hydrolysis equal to that of creatine-P and 2 kcal/mol greater than that of cyclocreatine-P. The relative rates of utilization of intracellular phosphagens by Ehrlich cells when their ATP pools were depleted with 2-deoxyglucose were in the order: creatine-P > N-ethylguanidinoacetate-P > cyclocreatine-P. Dietary N-ethylguanidinoacetate was nontoxic even at very high levels to all animal systems tested. Feeding of 2% N-ethylguanidinoacetate to mice or chicks resulted in equimolar replacement of natural by synthetic phosphagen to the following extents: heart, 75%; leg muscle, 50%; and brain 10–25%. N-Ethylguanidinoacetate-P is the most active synthetic phosphagen thus far found to be accumulated by animal tissues.     

Recent developments in the activation process of bovine chymotrypsinogen A

A brief account is given of the history, distribution, and activation events of proteins of the bovine chymotrypsinogen family. Recent developments in the investigation of the activation process of bovine chymotrypsinogen A are discussed, and a revised scheme for the overall activation process is presented.     

Developmental studies of lethality associated with the antennapedia gene complex in Drosophila melanogaster

A number of dominant homoeotic mutations are localized to the proximal right arm of chromosome 3 of Drosophila melanogaster and are thought to represent members of a gene complex that controls normal determinative decisions in the head and thorax. We have designated this complex the Antennapedia gene complex (ANT-C). Developmental studies were done to investigate the nature of the lethality associated with members of two of the complementation groups within ANT-C. The first complementation group, represented by the mutant Multiple Sex Combs (Msc) is characterized by embryonic lethality when heterozygous with a deletion of the ANT-C. The second complementation group consists of Antennapedia (Antp), Antennapedia-Extra Sex Combs (Antp^Scx), and the lethals recovered as revertants of Antp^Ns. When heterozygous for a deletion of the ANT-C or in heterozygous condition with each other, the members of this group show effective lethal phases spanning from embryo-larval boundary to late larval stages. Wakimoto and Kaufman (1981) show that the Antp⁺ gene acts to establish normal determinative states in the thorax. In the present work, transplantation of eye-antennal disks from lethal individuals heterozygous for two different Antp^Ns revertant chromosomes into wild-type hosts allowed the assessment of the function of the Antp⁺ allele in the antenna. Since these transplants formed only antennal structures and showed no evidence of the antennal → leg transformation seen in Antp^Ns controls, we conclude that the wild-type function of the Antp locus is not necessary for the establishment and/or maintenance of the antennal determined state. We suggest that regulatory mechanisms associated with the Antp⁺ structural gene normally function both to allow its expression in the thorax and to repress it in the antenna.     

Defects in embryogenesis in mutants associated with the antennapedia gene complex of Drosophila melanogaster

Embryogenesis in individuals with mutations or deficiencies of the genes in the polytene interval 84A-84B1,2 of Drosophila melanogaster was examined using scanning electron microscopy (SEM). The developmental function of this region of chromosome 3 is of particular interest since it contains the Antennapedia Gene Complex (ANT-C), a gene cluster that includes the homoeotic proboscipedia (pb), Sex combs reduced (Scr), and Antennapedia (Antp) loci. The results of SEM studies, clonal analyses, and temperature-shift experiments show that the fushi tarazu (ftz) and zerknullt (zen) genes, which map between pb and Scr, are involved in processes initiated during embryogenesis. The activity of ftz+ appears to be required within the first 4 hr of development for the establishment of the proper number of segments in the embryonic germ band. Individuals with ftz mutations or deficiencies produce only half the normal number of segments. Each of the segments is twice the normal width and is apparently comprised of cells that would normally form two separate metameres. The zen allele is required from about 2-4 hr of embryogenesis. Mutations of this gene result in disturbances of morphogenetic movements during gastrulation. The mutant phenotype is characterized by the absence of the optic lobe, defects in involution of the head segments, and in some cases, failure of germ band elongation. A requirement during embryogenesis for the activities of other genes residing in the 84A-84B1,2 polytene interval is suggested by the phenotypes of individuals heterozygous or homozygous for chromosomal deficiencies. Using the deficiencies Df(3R)AntpNs+R17, Df(3R)Scr, and Df(3R)ScxW+RX2, we examined the effects of deleting the distal portions or all of the 84A-84B1,2 interval. The defects in deletion heterozygotes suggest that the wild-type activity of some gene(s) other than zen, within or just adjacent to the 84B1,2 doublet, is required to complete normal head involution. The deletion of all the loci in the 84A5-84B1,2 interval results in grossly abnormal morphology and morphogenesis of the gnathocephalic appendages of the embryo. From these studies we conclude that mutations and deficiencies of genes associated with the ANT-C have profound effects on embryogenesis. The mutant phenotypes suggest, in addition to ensuring proper segment identity, the wild-type alleles of the 84A-84B1,2 genes are necessary for normal segmentation and elongation of the germ band and normal head involution.     

Switch in pattern formation after puncturing the anterior pole of Smittia eggs (Chironomidae, Diptera).

The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region.     

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.     

Leaf production and nitrogen and phosphorus tissue content of Littorella uniflora (L.) Aschers. In relation to nitrogen and phosphorus enrichment of the sediment in oligotrophic Lake Hampen,Denmark

Littoral sediments with a dense population of Littorella uniflora (L.) Ascers. were artificially enriched with nitrogen and phosphorus. Responses to increasing levels of nutrients were recorded as leaf production and tissue nutrient content. Phosphorus enrichment resulted in increasing P concentrations in all plant fractions (leaves, stem and roots), whereas only leaves and roots showed increasing N concentrations when inorganic N was added. Leaf production increased significantly after increasing the sediment P level 2, 3 and 5 times. Nitrogen did not affect leaf production. The tissue content of total N and P in control plants averaged 3.29 and 0.28%, respectively. Thus the critical P level in L. uniflora seems to be about 0.28%, which is higher than previously published values. It was also found that L. uniflora translocates phosphorus from old to younger leaves.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein

A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing. In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing. Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease. The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis. The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length. Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP. RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981). Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III. Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein. These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP. The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.     

Asynchronous division in cell colonies. I. General considerations and a linear control model

Asynchronous cell division is examined generally for features which have mathematical analogs in the field of statistical physics. A simple linear model for the controlling chemical kinetics is proposed, which yields the essential features of the experimental division time distribution. These results suggest an experiment by which the concept of talandic temperature (Goodwin, 1963) might be studied quantitatively.     

A specific inhibitor to δ-aminolevulinate dehydratase in rat bone marrow cells

A factor that specifically inhibited δ-aminolevulinate dehydratase was found in rat bone marrow cells. The inhibitor, which was located in the supernatant fraction of the bone marrow hemolysate, was purified about 12-fold by ammonium sulfate fractionation and column chromatography on Sephadex G-75. The partially purified inhibitor was heat labile and sensitive to trypsin and was denatured by urea. It had a pH optimum of 7.5–8.0, and a molecular weight of 28,000. It inhibited the activity of δ-aminolevulinate dehydratase noncompetitively.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

Crystal packing and stoichiometry of the fibre protein of adenovirus type 2

Crystals of the fibre protein of adenovirus type 2 have been grown and studied by electron microscopy and X-ray powder diffraction. The molecular packing and density of the crystals suggest that the fibre is dimeric.     

Purification of cytosolic,latent endoribonuclease from porcine thyroid

An alkaline endoribonuclease was purified 1800-fold from the cytosolic, latent ribonuclease fraction of porcine thyroids by gentle procedures specifically designed to exclude both heating and acidification steps. Polyacrylamide gel electrophoresis revealed a broad peak of enzyme activity that was coincident with the stained protein band. As estimated by gel filtration chromatography the major form of the enzyme (59%) had a molecular weight of 51,000; the remainder of the activity was distributed among six minor forms. Carboxymethyl-cellulose chromatography showed that the enzyme had at least three interconvertible forms. The latent alkaline ribonuclease had a pH optimum of 8.1 in both Tris and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffers and was stimulated by a number of monovalent chloride and potassium salts at ionic strengths between 10 and 70 mm; above 100 mm the salts were all inhibitory with the exception of ammonium chloride. At 1 mm both MgCl₂ and CaCl₂ were stimulatory, whereas CuCl₂ ZnCl₂ and EDTA were inhibitors. Both native and denatured DNA were slightly stimulatory. The porcine thyroid latent alkaline ribonuclease was specific for pyrimidine homopolymers and yielded a mixture of cyclic mononucleotides and oligonucleotides when incubated with poly(C). It did not hydrolyze 2′(3′)-cyclic CMP, purine homopolymers, native or denatured DNA or poly(A) · poly(U). Its activity toward rRNA was greater than toward tRNA and it cleaved the former to a mixture of mononucleotides and oligonucleotides. The properties of the intracellular, cytosolic, latent, alkaline ribonuclease distinguish it from pancreatic ribonuclease A and other nonsecretory ribonucleases.     

Reactivity of ferric Aplysia myoglobin towards anionic ligands in the acidic region: Proposal for a structural model

The equilibrium binding properties of ferric Aplysia myoglobin have been studied for a number of anionic ligands in the pH region from neutrality to ~4. For all the ligands studied, the intrinsic affinity of Aplysia metmyoglobin increases by more than one order of magnitude as the pH is lowered well below neutrality.The spectroscopic properties of the ligand-free and the ligand-bound molecules show a pH dependence with apparent pK values of 4.7 and 6.1, respectively.On the basis of temperature-jump experiments, a kinetic scheme has been proposed and rate constants have been measured for the binding of azide at pH 6 and pH 4.Kinetic and thermodynamic features match each other, suggesting that a single ionizing group is responsible for all the observed effects.By inspection of the three-dimensional structure, this group has been tentatively identified as the proximal imidazole. Protonation of the N_ε of proximal histidine would be associated to the rupture of the proximal bond, giving rise to the formation of a tetra-co-ordinate, ligand-free and penta-co-ordinate, ligand-bound molecule.     

Modification of the reactive sulfhydryl of bacterial luciferase with spin-labeled maleimides

Concerned about the lack of agreement among reports of the molecular weight of ferredoxin-NADP⁺ oxidoreductase, we carried out molecular weight determinations in the ultracentrifuge. Sedimentation velocity, sedimentation equilibrium, and active enzyme sedimentation studies all indicate that ferredoxin-NADP⁺ oxidoreductase in the purified form is active as a monomer of molecular weight 33,000–36,000.