Tryptic Peptide Patterns Research Articles

Enzyme evolution in the Enterobacteriaceae.

An immunological approach has been used for the study of alkaline phosphatase evolution in bacteria of the family Enterobacteriaceae. Antisera were prepared against alkaline phosphatase from Escherichia coli and Klebsiella aerogenes and tested against the unpurified alkaline phosphatases of 32 strains of enterobacteria by double diffusion and quantitative micro-complement fixation. The immunological relationships detected among the alkaline phosphatases of enterobacteria agree approximately with those reported for five other enzymes, as well as with the tryptic peptide pattern similarities found for two other enzymes, and with the relationships detected by interspecific deoxyribonucleic acid hybridization tests.

Changes in the primary structure of a mutationally altered ribosomal protein S4 of Escherichia coli.

Protein S4-su6 is a mutationally altered 30 S protein from Escherichia coli. S4-su6 suppresses the expression of streptomycin dependence controlled by another 30S protein. In addition, S4-su6 binds less tightly to its RNA binding site than the wild type form of S4. The present data show that at least seven amino acid replacements have occured in S4-su6. Moreover, the tryptic peptide pattern of S4-su6 is different from that of S4. Therefore, the pleiotropic effects of the mutation responsible for the altered structure of S4-su6 can be accounted for by extensive changes in the primary structure of S4-su6.

Subunit Structure of δ-Aminolevulinate Dehydratase from Mouse Liver

Abstract δ-Aminolevulinate dehydratase purified from livers of Swiss Webster mice has a molecular weight of 250,000 by sedimentation-diffusion, gel filtration chromatography, and centrifugation in sucrose gradients. Each mole of enzyme can bind 6 moles of substrate as shown by reduction of the intermediate Schiff base with NaBH4. Denaturation with 6 m guanidine hydrochloride or sodium dodecyl sulfate in the presence of reducing agents dissociates the native enzyme into subunits of 39,500 molecular weight. No NH2-terminal amino acid can be detected after reaction with dansyl chloride. There are 14 arginine and 12 lysine residues per 39,500 g of δ-aminolevulinate dehydratase polypeptide. Hydrolysis with trypsin yields approximately 27 peptides, 14 of which contain arginine. All evidence is consistent with mouse liver δ-aminolevulinate dehydratase being composed of six identical polypeptide chains. Alleles at the levulinate locus control the level of hepatic δ-aminolevulinate dehydratase by regulating the rate at which the enzyme is synthesized. Combined isotopic and immunochemical techniques reveal no differences in the subunit composition, number of active sites, or pattern of tryptic peptides between δ-aminolevulinate dehydratase from inbred strains of mice homozygous for different alleles at the levulinate locus. These results provide additional evidence that mutations of the levulinate locus do not affect the primary structure of hepatic δ-aminolevulinate dehydratase.

Serological and finger-printing analyses of mutant flagellar antigens of Salmonella.

SUMMARY: Spontaneous mutants with altered flagellar antigen were isolated from a phase-1 stable Salmonella strain, tr 6, whose phase-1 antigen consists of at least three antigenic factors, g3, g4 and f. Three kinds of antisera reacting with each of these factors were used as the selective agents. The mutants were isolated by picking from swarms in semisolid medium containing the respective antisera. Mutants were classified into two groups. A group comprising four mutants selected with antiserum to g3 and two mutants selected with antiserum to g4, in which all three antigenic factors examined were simultaneously altered; and a second group comprising all the other mutants, in which only a single factor corresponding to the selective antiserum was altered. All the altered factors retained weak cross-reactivity with the original factors. In all cross-absorption-agglutination tests between each of the mutant antigens and the wild-type antigen, activity of the antisera was fully absorbed, showing that the mutant antigens had neither lost any antigenic specificities nor gained any new specificity. Transductional analysis showed that the sites of mutation were inseparable from H 1, the structural gene for phase-1 flagellin. Tryptic peptide patterns of flagellins of two mutants were compared with that of the wild-type strain and a difference of one or two peptides was detected. From these results it is concluded that these mutants possessed a minor change in primary structure of flagellin which resulted from a mutation in the H 1 gene.

Evidence for a second haemoglobin alpha-locus duplication in Macaca irus.

IN a study1 of the haemoglobins of Macaca irus obtained from Thailand and Vietnam two α-chain variants, electrophoretically distinguishable from the normal type (Hb-Ami), were found to be frequent. One, Hb-Qmi, was anodal to Hb-Ami on starch gel at pH 8.6 and varied considerably in relative concentration when present with the latter. The fingerprint pattern of the soluble tryptic peptides was, however, indistinguishable from that of Hb-Ami and the difference is presumably in the insoluble peptide core. The other variant, Hb-Pmi, had the same electrophoretic mobility as Hb-Qmi in very fresh haemolysates but formed aggregates with ageing so that it then appeared in starch gels as a trail extending cathodally from the Hb-Ami position. The fingerprint of Hb-Pmi was distinct from that of Hb-Ami, αT3 (tryptophan positive) and αT4 (histidine positive) being absent and a new tryptophan and histidine positive peptide appearing in the neutral region. A single amino-acid substitution in α15 or 16 would probably account for this. The phenotype frequencies of these two variants in the population were consistent with their being determined by alleles at the α-locus. In addition, about 20 per cent of the animals had a minor haemoglobin component. The distinctive chain of this component was shown2,3 to differ from the normal α-chain in at least four substitutions. In some animals the minor component was present together with Hb-Ami and either Hb-Pmi or Hb-Qmi and it was suggested that the characteristic chain arose by duplication of the α-locus and mutational divergence.

Multiple hemoglobins in the golden hamster

1. 1. Seven distinct benzidine-positive bands were found on starch-gel electrophoresis at pH 8.6 of the hemolysates from fetal, newborn and adult golden hamsters. Four of the seven benzidine-positive bands were analyzed and named Hemoglobins I, II, III and IV according to their increasing electrophoretic mobility. Hemoglobins I and II were found as major components in the fetal stage. Hemoglobin I gradually decreased and disappeared by the 11th day after birth. Hemoglobin II, however, gradually decreased but remained as a minor component in the newborn and adult. Hemoglobins III and IV were found as minor components in the fetal stage, the former as a major component and the latter as a minor one in the newborn and adult stages. 2. 2. The amino acid compositions of the polypeptide chains from these hemoglobins were determined. Few differences among the α I, α III and α IV chains were found in the amino acid compositions. There were apparent differences between the α II and other α chains involving a minimum of eleven amino acid residues. Among the non-α chains, the β II chain was very similar to the β III chain and there were distinct differences among the γ I, β III and β IV chains. 3. 3. The tryptic peptide patterns of the aminoethylated (AE) α I, AE α II and AE α IV chains were closely similar to one another. In the patterns of the AE α III and AE α II chains, one peak of the AE α III was replaced by one acidic peak and two basic peaks of the AE α II chain. On the other hand the AE γ I, AE β III and AE β IV chains were quite different from one another in the tryptic pattern, while the pattern of the AE β II chain was similar to that of the AE β III chain.

Isolation of an arginine-rich protein from particles of adenovirus type 2

Polyacrylamide gel electrophoresis showed that particles of adenovirus type 2, disrupted with hot sodium dodecyl sulphate (SDS) and dithiothreitol, contained nine protein components (bands 1–9) with molecular weights ranging from 190,000 to 11,000. Four of these components (bands 6, 7, 8 and 9) were isolated by preparative acrylamide gel electrophoresis, following a preliminary fractionation of the proteins by stepwise ethanol precipitation in the presence of guanidine hydrochloride. One component (band 2) was isolated by the latter procedure alone. The protein in band 8, one of the core proteins of the virus, was found to contain 23% of arginine. It also had an unusually high content of alanine (20%). This very arginine-rich protein had a molecular weight of approximately 18,000 and possessed a free N-terminal alanine residue. The protein in band 2 (which was isolated solely by ethanol fractionation in the presence of guanidine hydrochloride) had a molecular weight of approximately 120,000. It probably contained only the hexon protein of the virus, as it had the same amino acid composition and showed the same pattern of tryptic peptides as the hexon of adenovirus type 2 crystallized from the soluble antigen fraction of infected cell extracts.

Further evidence on the formation of poliovirus proteins

When poliovirus-specific polypeptides are synthesized in the presence of the amino acid analogs, canavanine, p-fluorophenylalanine, and azetidine-2-carboxylic acid, a very large polypeptide, NCVP-00, not ordinarily found in infected cells, is produced. The molecular weight of this polypeptide exceeds 200,000 daltons and it may, therefore, represent the entire informational content of poliovirus RNA. In experiments where amino acid analogs are not present, no nascent polypeptides larger than about 130,000 daltons can be detected. This implies that ordinarily some proteolytic cleavages occur before the NCVP-00 molecule is completely synthesized. These cleavages and others which occur more slowly can be partially inhibited by diisopropyl fluorophosphate. Structural similarities between different poliovirus-specific polypeptides have been investigated by chromatography of tryptic hydrolysates of pairs of differentially-labeled proteins. These experiments, corroborating previous results, suggest that the procapsid protein, VP-0, is cleaved into two of the virion proteins, VP-2 and -4. Furthermore, all of the procapsid proteins appear to be cleavage products of one large precursor, NCVP-1, which has a molecular weight of 125,000 daltons. Analysis of tryptic peptide patterns has also confirmed the existence of a non-structural polypeptide, NCVP-X, which has the same electrophoretic mobility as a virion protein, VP-1.

The Apparent Microheterogeneous Nature of the High-Sulfur Proteins of α-Keratins

The homogeneity of wool SCMK-B2 and mohair SCMK-B2, prepared according to the procedure of Gillespie, has been investigated. Moving boundary electrophoresis and gel filtration of the preparations revealed a single peak. Examination of the SCMK-B2 preparations by disc electrophoresis demonstrated the presence of various components. The preparations were each separated by chromatography on DEAE-cellulose into four subfractions. The aminoacid composition and tryptic peptide patterns of the subfractions were similar but certain characteristic differences were obvious.

A Comparison of the Tipula and Sericesthis Iridescent Viruses

Summary A direct experimental comparison of the Tipula iridescent virus and the Sericesthis iridescent virus has shown them to be very similar but not identical. After extensive purification the viruses were differentiated by electrophoretic mobility, tryptic peptide patterns, serological cross-reactivity, and pathogenicity in a group of host insects. The viruses were identical in gross chemical composition and in a number of physical properties. A particle weight of 1.2 to 1.3 × 109 daltons was calculated by three methods. A slower sedimenting top component consisting of empty protein shells was found during the purification of each virus; similar empty shells could be produced by succinylation or formic acid-diphenylamine treatment of whole virus.

A study of the coat proteins of bacteriophage ∅X174

Three protein components associated with purified ∅X174 particles have been separated by means of preparative paper electrophoresis in formic acid. These components, here called α, β, and γ, constitute approximately 15–20%, 50–60%, and less than 3%, respectively, of the mass of the purified bacteriophage particles. α, β, and γ differ in their amino acid composition and tryptic peptide pattern. N-terminals methionine and serine were detected in the coat proteins of bacteriophage ∅X174, in a molar ratio of methionine:serine = 1:3. The N-terminal amino acid of protein α has been found to be methionine.

The subunits of bovine liver glutamate dehydrogenase: Demonstration of a single peptide chain

A complete amino acid analysis is presented for bovine glutamate dehydrogenase. Dinitrophenyl and phenylthiohydantoin end-group analysis suggests that this protein consists of polypeptide chains containing alanine as the only amino terminal residue. Threonine was identified as the C-terminal residue by hydrazino-lysis and carboxypeptidase digestion, and quantitative estimation showed close to one C-terminal residue per mole of protein weighing 52,000. These results, along with analysis of the tryptic and cyanogen bromide peptide patterns, support the view that glutamate dehydrogenase is composed of very similar or identical subunits.

Lobster hemocyanin: properties of the minimum functional subunit and of aggregates.

Lobster hemocyanin dissociates into a functional subunit of 68,000 to 70,000 molecular weight when Ca(2+) ions are removed from an alkaline solution of low ionic strength. Succinylation results in a further dissociation into two nonfunctional subunits of approximately 34,000 to 35,000 molecular weight. Amino acid analysis and tryptic peptide patterns indicate that the functional subunit is composed of at least two polypeptide chains which are similar.

The subunit polypeptides of human fibrinogen

Three polypeptide chains have been isolated from S-sulfofibrinogen by chromatography on carboxymethylcellulose. The three chains, designated A, B, and C, were pure as judged by ultracentrifugal analysis and zone electrophoresis. Each chain corresponded to one of the three electrophoretic components observed in unfractionated S-sulfofibrinogen. By the methods of sedimentation-equilibrium analysis in the ultracentrifuge, the molecular weights of the chains were estimated to be 47,000 for the A-chain, 56,000 for the B-chain, and 63,500 for the C-chain. The amino acid composition and tryptic peptide pattern of each chain was unique. If it is assumed that human fibrinogen contains one pair of each of the unique chains, then the molecular weight and amino acid composition of fibrinogen are closely accounted for by the weights and composition of the chains.

TRYPTIC PEPTIDE PATTERNS OF SOME MAMMALIAN HEMOGLOBINS.

Journal of Cellular and Comparative PhysiologyVolume 63, Issue 1 p. 1-6 Article Tryptic peptide patterns of some mammalian hemoglobins Charles W. Foreman, Charles W. Foreman Pfeiffer College, Misenheimer, North Carolina and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Address after September 1, 1963: Department of Biology, University of the South, Sewanee, Tennessee.Search for more papers by this author Charles W. Foreman, Charles W. Foreman Pfeiffer College, Misenheimer, North Carolina and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Address after September 1, 1963: Department of Biology, University of the South, Sewanee, Tennessee.Search for more papers by this author First published: February 1964 https://doi.org/10.1002/jcp.1030630102Citations: 11 Operated by Union Carbide Corporation for the United States Atomic Energy Commission. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume63, Issue1February 1964Pages 1-6 RelatedInformation

Amino-Acid Composition of the Polypeptide Chains of Gorilla Hæmoglobin

COMPARISON of tryptic peptide patterns has demonstrated the great overall similarity of the primary structure of haemoglobins from normal adult man and anthropoid apes1. Work is now in progress to define this similarity more precisely. We report here the determination of the amino-acid composition of the polypeptide chains of gorilla haemoglobin.

A COMPARISON OF ANIMAL HEMOGLOBINS BY TRYPTIC PEPTIDE PATTERN ANALYSIS

The complete amino acid sequences (primary structure) of hemoglobins can, in principle, be determined by methods currently available. Although detailed studies of the primary structure of human and horse hemoglobins are in progress in several laboratories,(1) the methods are so laborious that complete sequences have not yet been established. Important questions in the realm of genetics and evolution require the immediate examination of the structure, primary and other, of many different hemoglobins. The application of methods that are quicker, though less informative and reliable, than the techniques required for complete sequence determination is therefore in order as a provisional means of securing useful information. Such a method is the analysis of peptide patterns obtained by combined paper electrophoresis and chromatography of tryptic hydrolysates of denatured hemoglobin.(2) Of particular interest are comparisons between hemoglobin components present in (a) organisms of one animal species at a given time in development, (b) organisms of one species at different stages of development, and (c) organisms of different species. The present paper is concerned exclusively with the last type of comparison. In order to scan the range of variation of hemoglobin structure throughout evolution, hemoglobins from a number of animals both closely and distantly related to man have been selected and compared as to tryptic peptide patterns with human hemoglobin A. Whole hemoglobin preparations from adult animals have been studied throughout. The problem of individual heterogeneity will be treated elsewhere.