Showing papers on "Peptide sequence published in 1977"

Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone

Abstract: The primary structure of DNA containing the sequence for rat pituitary growth hormone mRNA has been determined. DNA was obtained by reverse transcription of polyadenylated RNA from cultured pituitary cells and from recombinant bacterial plasmids. The amino acid sequences for rat growth hormone and its precursor form have been deduced from the determined nucleotide sequences.

Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24.

Abstract: Chromosomal protein A24 has a unique structure inasmuch as it contains histone 2A and a nonhistone polypeptide the sequence of which has been partially determined. Comparative analysis of the ninhydrin-insensitive amino-terminal tryptic peptides of protein A24 and histone 2A and a quantitative analysis of their carboxyl-terminal amino acid indicated that protein A24 has two amino termini and one carboxyl terminus. The amino acid sequence analysis of tryptic peptide 17' of protein A24: (see text) showed it contains tryptic peptide 17 of histone 2A, Lys-Thr-Glu-Ser-His-His-Lys. Lysine 119, the amino terminus of this peptide, which is derived from the histone 2A portion of protein A24, is linked by an isopeptide bond to the carboxyl group of a glycine residue. Accordingly, the branched structure of protein A24 proposed is: (see text).

Characterization of C-reactive protein and the complement subcomponent C1t as homologous proteins displaying cyclic pentameric symmetry (pentraxins)

Abstract: Partial amino acid sequences of rabbit C-reactive protein, a peptide derived from human C-reactive protein by cyanogen bromide cleavage, and the C1t subcomponent of the human complement component C1 have been determined. Extensive sequence homology between these proteins establish their evolutionary relationships. In addition, examination of C-reactive proteins by negative-stain electron microscopy revealed that the protein is composed of five subunits arranged in cyclic symmetry. This structure is similar to that reported for both C1t and the amyloid P-component. The extensive structural relationship suggests similar or overlapping functions and the term pentraxin is proposed to describe these homologous proteins.

Characterization of a common precursor to corticotropin and beta-lipotropin: identification of beta-lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system.

Abstract: Radioactive proteins synthesized in an mRNA-dependent reticulocyte cell-free system under the direction of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either alpha(1-24) corticotropin or beta-endorphin [beta(61-91) lipotropin]. Each immunoprecipitate was fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and shown to contain only one labeled protein with an apparent molecular weight of 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin and beta-lipotropin molecules isolated from the gels demonstrated that the two proteins had the same lysine, methionine, and tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromatographic mobilities as marker tryptic peptides from bovine beta-melanotropin and porcine beta-endorphin. The identification of these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The beta-lipotropin tryptic peptides were also shown to be located carboxy terminal to the corticotropin tryptic peptides.

Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide.

Abstract: The amino acid sequence of a biologically active polypeptide isolated from calf thymus, termed thymosin alpha1, has been determined. Thymosin alpha1 is a heat stable, highly acidic molecule composed of 28 amino acid residues. This peptide is one of several present in thymosin fraction 5 that may participate in the regulation, differentiation and function of thymus-dependent lymphocytes (T cells). A nomenclature for the family of polypeptides present in thymosin fraction 5 is suggested.

Amino acid sequence of human platelet factor 4

Abstract: Human platelet factor 4, a protein that binds heparin, has been purified to apparent homogeneity and the complete amino acid sequence of the protein has been determined. The 70-residue polypeptide chain contains no methionine, tryptophan, or phenylalanine, and contains only a single tyrosyl residue. The sequence analysis demonstrates a highly negatively charged amino-terminal region. The carboxyl-terminal region of the polypeptide is unusual in that it contains a repetitive clustering of positively charged and hydrophobic pairs of amino acids; preliminary evidence suggests that this domain may play a role in the binding of heparin.

Cleavage at aspartyl-prolyl bonds.

Abstract: Specific cleavage at AspPro bonds of a protein can be effected by exposure to acid at moderate temperature for periods up to 120 hr. The effectiveness of cleavage is usually increased by incorporating a denaturing agent in the acid solution. Some AspPro bonds are resistant to cleavage probably because of retention of protein folding even under the most vigorous conditions that have been employed, and under these circumstances some nonspecific peptide bond cleavage may occur, particularly if the exposure is prolonged. It is very likely that cleavage at AspPro bonds will occur when acid procedures are employed in peptide bond scission or peptide separation and purification. With the larger peptides produced by cyanogen bromide cleavage it has been observed that partial cleavage at AspPro near the termini of the authentic peptide may yield fragments that copurify with the larger component, thus complicating subsequent sequence determinations.13 The relative paucity of AspPro bonds in proteins is both a limitation and an advantage of this specific cleavage procedure. The limitation is that many proteins do not contain any AspPro bonds but as the AspPro bond is never abundant the number of fragments obtained by application of the procedure can be advantageously small, which will simplify subsequent fractionation processes.

Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane.

Abstract: The messenger RNA for the lipoprotein of the E coli outer membrane was found to code for a putative precursor, prolipoprotein, which has 20 additional amino acid residues extending from the amino terminus of the lipoprotein Using the prolipoprotein synthesized in an E coli cell-free system directed by purified messenger RNA for the lipoprotein, the complete amino acid sequence of the amino-terminal precursor region was determined to be as follows: (formula: see text) It was also found that the prolipoprotein that accumulates in toluene-treated cells has the same sequence The significance of the amino acid sequence is discussed in terms of the mechanism of biosynthesis and assembly of the lipoprotein in the E coli outer membrane

Cleavage at glutamic acid with staphylococcal protease.

Abstract: Publisher Summary Proteolytic enzymes catalyzing the hydrolysis of peptide bonds involving exclusively the basic amino acid residues lysine and arginine have been available for many years. Trypsin is by far the best known enzyme exhibiting this high degree of specificity and, for that reason, it has played a central role in the studies of the primary structure of proteins. Recently, enzymes that specifically cleave peptide bonds at the carboxyl group of the acidic amino acid residues, aspartic acid and glutamic acid, have been discovered. One of these enzymes, staphylococcal protease, has this specificity and can be further restricted to glutamyl bonds only under certain controlled conditions. This enzyme can be used for the determination of the amino acid sequences of several proteins and proved to be another valuable tool for such studies. Staphylococcal protease shows a marked preference for certain aspartyl bonds when used in ammonium bicarbonate or acetate buffer. The staphylococcal protease is fully active in the presence of 0.2% sodium dodecyl sulfate and retains 50% of its activity in a 4 M urea solution. Digestion under these conditions could be attempted for proteins or peptides that are not readily attacked by the protease under nondenaturing conditions.

Repetitive sequences in protein A from Staphylococcus aureus. Arrangement of five regions within the protein, four being highly homologous and Fc-binding.

Abstract: After tryptic digestion of intact Staphylococcus aureus the residual portion of protein A that was still bound to the cell wall was cleaved off with lysostaphin. From the two digests Fc-binding fragments were isolated and the following characteristics observed. (a) There are four Fc-binding, highly homologous regions, each consisting of 58--62 amino acid residues. (b) These regions are consecutively arranged from the N-terminal part of the protein. (c) The residual C-terminal part, approximately 150-residues long, differs to a great extent with respect to primary and secondary structures from the four active regions. Furthermore, it is suggested that the protein is bound to the bacterial cell wall structure through this C-terminal part.

Complete amino acid sequence of rabbit muscle glycogen phosphorylase.

Abstract: The sequence of the 841 amino acid residues in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy) which were fragmented by cleavage at methyonyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder of the sequence. The residues involved in the covalent and allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679, respectively.

Primary structure of human C-reactive protein.

Abstract: The complete amino acid sequence of human C-reactive protein has been established. Distant homologies to C3 homology region in the CH2 domain of IgG and to C3a anaphylotoxin have been noted. No homology to other immunoglobulin homology regions or to the same homology region in other heavy chains was observed. The previously reported homologies between rabbit and human C-reactive protein and protein C1t have been extended and strengthened.

Activation of lipoprotein lipase by native and synthetic fragments of human plasma apolipoprotein C-II.

Abstract: Apolipoprotein C-II (apoC-II), a protein constituent of human very low density lipoproteins, is the activator for lipoprotein lipase (LPL; triacylglycerol acyl-hydrolase, EC 3.1.1.3). The amino acid sequence of the 78 residues of apoC-II has recently been established in this laboratory. To determine the minimal sequence requirements for activation, we have prepared both native and synthetic fragments of apoC-II and tested them for their ability to activate LPL. Cyanogen bromide fragments of apoC-II corresponding to residues 1--9 and 10--59 had little ability to activate LPL. However, the COOH-terminal cyanogen bromide fragment corresponding to residues 60--78 increased hydrolysis 4-fold compared to an average of 9-fold activation for the same concentration of apoC-II. The synthetic peptide containing residues 60--78 prepared by solid-phase techniques enhanced the lipolysis 3-fold. Addition of five residues produced a synthetic fragment 55--78 that enhanced the release of fatty acid 12-fold compared to 13-fold for intact apoC-II. By contrast, the synthetic peptide containing residues 66--78 did not activate. Removal of the three COOH-terminal residues, Gly-Glu-Glu, from fragment 60--78 decreased the ability to activate LPL by greater than 95%. These studies suggest that the maximal activation of LPL by apoC-II requires a minimal sequence contained within residues 55--78.

The amino acid sequence of beta-galactosidase of Escherichia coli.

Abstract: The amino acid sequence of beta-galactosidase was determined. The protein contains 1021 amino acid residues in a single polypeptide chain. The subunit molecular weight calculated from the sequence is 116,248. The sequence determination, carried out mainly by conventional methods, was aided by complementation tests, by the use of termination mutant strains, and by a new immunochemical method. The five residue sequence Thr-Pro-His-Pro-Ala appears twice within the polypeptide chain, but no other striking homologous features are evident.

Penicillopepsin from Penicillium janthinellum crystal structure at 2.8 A and sequence homology with porcine pepsin.

Abstract: The polypeptide chain of the acid protease penicillopepsin folds via an 18-stranded mixed β-sheet into two distinct lobes separated by a 30-A long groove which is the extended substrate binding site. The catalytic residues Asp-32 and Asp-215 are located in this groove and their carboxyl groups are in intimate contact. Alignment of the amino acid sequence with that of pepsin shows regions of high homology.

Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen.

Abstract: M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

Serum albumin: recent progress in the understanding of its structure and biosynthesis.

Abstract: Major discoveries have been made in the past few years on the structure and mode of biosynthesis of serum albumin. The complete amino acid sequence of this protein has been determined, and its covalent structure shown to be a single peptide chain grouped into a series of nine disulfide-bonded loops. These loops appear to associate into three similar domains. By study of isolated fragments of the molecule it can be demonstrated that the binding of billirubin and the primary binding of long-chain fatty acids are functions of separate domains. The biosynthesis of albumin has been found to involve a precursor form, termed "proalbumin", in which a basic hexapeptide is attached to the amino end of the chain. Similar precursor forms are now known to have a role in the formation of other secreted proteins, but in the case of albumin the purpose of the additional peptide is not clear. Clinical methodology for albumin assay has advanced but little despite--or perhaps in part because of--the increasing use of automation. Hope for improvement is foreseen in the advent of immunochemical procedures and in a better understanding of the specificity of dye-binding reactions.

Cleavage of Rous sarcoma viral polypeptide precursor into internal structural proteins in vitro involves viral protein p15.

Abstract: The polypeptide precursor pr76 to the internal viral group specific (gs) antigen proteins of Rous sarcoma virus, synthesized in a cell-free system of ascites cells, has been processed in vitro into the viral proteins by purified viral protein p15 as well as by disrupted Rous sarcoma virus. Disrupted Rauscher murine leukemia virus does not stimulate the cleavage process in vitro. Autocatalytic cleavage of the polypeptide precursor pr76 or Rous sarcoma virus, which contains the peptide sequence of p15, is not observed.

Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins

Abstract: A new technique is described to identify antigenically related proteins by radioimmunoassay after sodium dodecyl sulfate/polyacrylamide gel fractionation. When adult mouse myelin was examined by this technique, four proteins that are antigenically related to the small myelin basic protein were identified. They were designated: prelarge (molecular weight 21,500), large (18,500), presmall (17,000), and small (14,000). The four proteins were isolated by elution from polyacrylamide gels, and each protein migrated as a single band when analyzed by either sodium dodecyl sulfate or acidic polyacrylamide gel electrophoresis. Serial dilutions of the purified proteins were measured by radioimmunoassay. Both the slope of the inhibition curve and the level of maximal inhibition for each protein were the same as for the small myelin basic protein, indicating that each of the four proteins contains all of the antigenic sites present in the small basic protein. Structural relationships among the four proteins were examined by using two-dimensional analysis of tryptic digests. The results showed that: large was similar in amino acid sequence to the major myelin basic protein from other species; small was identical in sequence to large, except for an internal deletion of approximately 40 amino acid residues: prelarge contained the sequence of large plus an additional sequence of 25-35 amino acid residues; and presmall contained the sequence of small plus the same additional sequence as in prelarge. The four proteins were also treated with 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolienine (BNPS-skatole) which cleaves proteins specifically at tryptophan residues. Analysis of the cleavage products indicated that the additional amino acid sequence in both prelarge and presmall extends from the amino terminus of the molecule. Several implications of these results are discussed.

Primary structure of very low density apolipoprotein C-II of human plasma.

Abstract: Apolipoprotein C-II (apoC-II), a protein constituent of very low density lipoproteins of human plasma and the activator protein of lipoprotein lipase, has been isolated and its amino acid sequence has been studied. The protein has 78 amino acid residues and is lacking cysteine, cystine, and histidine. Chromatography on Bio-Gel P-30 in 25% formic acid of the cyanogen bromide digest of apoC-II yields three fragments designated as CNBr-I, -II, and -III. They contained 50, 19, and 9 residues, respectively. The alignment of the cyanogen bromide fragments has been established as CNBr-III-I-II by isolation and sequence of the tryptic peptides of the intact protein. The amino acid sequences of the tryptic and CNBr peptides were determined by conventional methods. With this information, it was possible to establish the complete amino acid sequence of apoC-II.

Purification and structural characterisation of human HLA-linked B-cell antigens

Abstract: The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.