Showing papers on "Protein primary structure published in 1978"

Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence.

Abstract: Peptides of glycophorin AMN were prepared by cyanogen bromide cleavage and by chymotryptic and tryptic digestion. Cyanogen bromide cleavage produces three fragments which account for the entire polypeptide chain. Trypsin and chymotrypsin cleave completely at several sites, but incompletely at sites within the glycosylated segment of the polypeptide chain. Some of the latter sites become accessible to proteolysis after desialation in addition to exposure of new sites for cleavage. The amino acid sequence of glycophorin AMN has been determined by manual Edman degradation, using both the direct Edman and the dansyl-Edman procedures simultaneously for determination of glycosylated amino acid residues. The automated procedure was used for sequence determination of a hydrophobic peptide. Glycophorin A is a polypeptide chain of 131 amino acid residues and contains 16 oligosaccharide units attached to the amino-terminal third of the molecule. Fifteen oligosaccharides are linked O-glycosidically to either threonine or serine residues and one complex oligosaccharide unit is attached N-glycosidically to an asparagine residue. Amino-terminal sequences are different for glycophorin AM and AN, the two forms of the glycophorin A molecule coded for by genes at the MN locus. The differences in sensitivity to proteases of various sites on glycophorin A seem to be due to heterogeneity in the carbohydrate components and not to differences in the primary structure of the polypeptide chains. This work contains a number of revisions and corrections of earlier preliminary reports [Segrest, J.P., Jackson, R. chem. Biophys. Res. Commun, 49, 964-969; Tomita, M., & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968].

Complete covalent structure of human beta-thromboglobulin.

Abstract: The complete primary structure of the platelet-specific protein human beta-thromboglobulin has been determined. beta-Thromboglobulin consists of identical subunits of 81 amino acids, each with a molecular weight of 8851. The amino acid sequence of the beta-thromboglobulin subunit is: Gly-Lys-Glu-Glu-Ser-Leu-Asp-Ser-Asp-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr-Thr-Ser-Gly-Ile-His-Pro-Lys-Asn-Ile-Gln-Ser-Leu-Glu-Val-Ile-Gly-Lys-Gly-Thr-His-Cys-Asn-Gln-Val-Glu-Val-Ile-Ala-Thr-Leu-Lys-Asp-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Pro-Asp-Ala-Pro-Arg-Ile-Lys-Lys-Ile-Val-Gln-Lys-Lys-Leu-Ala-Gly-Asp-Glu-Ser-Ala-Asp. Disulfide bridge-18 to half-cystine-58. The amino acid sequence of beta-thromboglobulin shows a marked homology with that of platelet factor 4. When the sequences are aligned for maximum homology, 42 of the 81 residues of beta-thromboglobulin are identical with those of platelet factor 4, including the position of the four half-cystines.

The amino acid sequence of Physarum actin.

Abstract: THE realisation that actin is involved not only in contractility in muscle tissue but also in general cytoplasmic movement and cell locomotion was based on the isolation of actin from lower eukaryotic organisms, such as Physarum polycephalum1, which show active protoplasmic streaming. It is now accepted that actin is the major structural protein of microfilaments characteristically found in the cytoplasm of most if not all higher cells, and that actins have been highly conserved during eukaryotic evolution and differentiation due to the constraints imposed by the large number of specific interactions which actin shows with other cytoplasmic proteins (for a review see ref. 2). Preliminary fingerprint analysis on Physarum actin3 and partial amino acid sequence analysis on actin from Acanthamoeba (ref. 4 and Elzinga cited in ref. 2) have emphasised this point, although so far a complete amino acid sequence has not been reported for actin from a lower eukaryotic species. Our studies on the primary structure of different mammalian actins have shown that several cytoplasmic actins are very similar if not identical in amino acid sequence, although they differ by at least 25 amino acids from the corresponding skeletal muscle actin5. The availability of fast amino acid sequence procedures for actin5 led us to study the actin from Physarum polycephalum to assess how cytoplasmic actins differ between lower and higher eukaryotes. We report here results which show that Physarum plasmodia, in contrast to non-muscle cells of higher vertebrates5–7, contain only one cytoplasmic actin species. This actin is more closely related to mammalian cytoplasmic actins than these actins are to skeletal muscle actin. The high degree of structural conservation of the actin sequence and structure during eukaryotic evolution is illustrated by the absence of any amino acid deletions or additions to the polypeptide chain beyond residue 2. Furthermore, the distribution of the proline residues remains unchanged and substitutions of amino acid residues with charged side chains are very rare, indicating a high conservation of the surface topography of the actin molecule.

The Primary Structure of Bovine Pancreatic Phospholipase A2

Abstract: The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.

Primary structure of the lambda repressor.

Abstract: The complete covalent structure of the bacteriophage lambda repressor has been determined by sequential Edman degradation, gas chromatographic-mass spectrometric peptide sequencing, and DNA sequencing of the repressor gene cI. The repressor is a single-chain, acidic protein containing 236 amino acids. The amino terminal 40 residues are highly polar and basic. Lysines and arginines in the sequence tend to be clustered.

Free ubiquitin is a non-histone protein of trout testis chromatin

Abstract: A NON-HISTONE chromosomal protein extracted from trout testis chromatin by 0.35 M salt, protein S, (ref. 1) has now been purified to homogeneity and its primary structure determined (Fig. 3) and shown to be identical with that of the thymus protein, ubiquitin2–4 (originally called thymopoietin) and sequenced by Schlesinger et al.5. Ubiquitin was previously reported in chromatin by Goldknopf et al.6 as a component of protein A-24 in which it is covalently bound by the α-carboxyl group of its C-terminal glycine to the e-amino group of lysine 119 of histone H2A in an isopeptide linkage7. Goldknopf and Busch8 have reported that protein A-24 is a component of the core nucleosomes of rat liver chromatin being present in quantities sufficient for it to be linked to H2A in 10–20% of the total nucleosomes. The present studies indicate clearly that ubiquitin can also occur as a free polypeptide in trout testis chromatin and is readily extracted by 0.35 M NaCl. Moreover, free trout testis ubiquitin (protein S) is rapidly released together with HMG-T (ref. 9) (and small amounts of Hl) during limited micrococcal nuclease digestion of trout testis chromatin and is probably also located in the linker regions of the chromatin. DNase I digestion does not release free ubiquitin preferentially10. In view of the pronounced biological effects of free ubiquitin on membrane adenyl cyclase of immature thymocytes3,4 and the ability of ubiquitin to promote the differentiation of these cells into mature T cells3,4, its occurrence in chromatin raises the possibility of a role of ubiquitin in cell differentiation and differential gene expression.

Repeating covalent structure of streptococcal M protein.

Abstract: We have attempted to identify the covalent structure of the M protein molecule of group A streptococci that is responsible for inducing type-specific, protective immunity. M protein was extracted from type 24 streptococci, purified, and cleaved with cyanogen bromide. Seven cyanogen bromide peptides were purified and further characterized. Together, the peptides account for the entire amino acid content of the M protein molecule. Each of the purified peptides possessed the type-specific determinant that inhibits opsonic antibodies for group A streptococci. The primary structures of the amino-terminal regions of each of the purified peptides was studied by automated Edman degradation. The partial sequences of two of the peptides were found to be identical to each other and to that of the uncleaved M protein molecule through at least the first 27 residues. The amino-terminal sequences of the remaining five peptides were identical to each other through the twentieth residue but completely different from the amino-terminal region of the other two peptides. However, the type-specific immunoreactivity and the incomplete analysis of the primary structure of the seven peptides suggest that the antiphagocytic determinant resides in a repeating amino acid sequence in the M protein molecule.

The complete amino-acid sequence of histone H2B(3) from sperm of the sea urchin Parechinus angulosus.

Abstract: The primary structure of a third H2B histone isolated from sperm of the sea urchin Parechinus angulosus has been determined. H2B(3) consists of a polypeptide chain of the following 148 amino acid residues: Pro-Arg-Ser-Pro-Ala-Lys-Thr-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Ser-Arg-Lys-Ala-Ser-Pro-Lys-Arg-Gly-Gly-Lys-Gly-Ala-Lys-Arg-Ala-Gly-Lys-Gly-Gly-Arg-Arg-Arg-Arg-Val-Val-Lys-Arg-Arg-Arg-Arg-Arg-Arg-Glu-Ser-Tyr-Gly-Ile-Tyr-Ile-Tyr-Lys-Val-Leu-Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Arg-Ala-Met-Ser-Val-Met-Asn-Ser-Phe-Val-Asn-Asp-Val-Phe-Glu-Arg-Ile-Ala-Ser-Glu-Ala-Ser-Arg-Leu-Thr-Ser-Ala-Asn-Arg-Arg-Ser-Thr-Val-Ser-Ser-Arg-Glu-Ile-Gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His-Ala-Val-Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr-Lys-Tyr-Thr-Ser-Arg. H2B(3)Parechinus closely resembles H2B(2)Parechinus but has one additional repeating pentapeptide in the amino-terminal region and a serine replacing glycine at position 98.

The primary structure of staphylococcal protease.

Abstract: The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protea...

beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

Abstract: A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Amino acid sequence of progesterone-induced rabbit uteroglobin.

Abstract: Uteroglobin, a steroid-binding protein of the uterine secretion of the rabbit which is induced by progesterone, comprises two identical polypeptide chains of 70 amino acid residues linked by two disulfide bonds. The primary structure has been determined by using both automated and manual methods of Edman degradation. Overlapping peptides were isolated from tryptic, and CNBr digests. The sequence is not homologous to any known protein except for a small acidic region (residues 22-29) resembling a sequence found in somatotropin. The C-terminal half is relatively basic. Implications for the secondary structure are discussed.

Common ancestor for concanavalin A and lentil lectin

Abstract: The primary structure of the alpha subunit from Lens culinaris lectin was determined by analysis of tryptic peptides and was shown to consist of 52 amino acid residues. The molecular weight calculated on the basis of the sequence is 5928. The whole chain is homologous with the region between positions 75 and 121 from concanavalin A. The NH2-terminal sequence of the beta chain, determined by automated Edman degradation, is homologous to another portion of the concanavalin A molecule, between positions 123 and 165. Comparison of the 94 residues from the lentil lectin alpha and beta chains with concanavalin A reveals the existence of 43 identities. Thirty-four other homologies could have arisen, each by a single nucleotide substitution. This extensive homology suggests that the lentil lectin alpha and beta chains may be proteolytic fragments from a single polypeptide chain of the same length as concanavalin A.

µ-Chains from a Nonsecretor B Cell Line Differ from Secreted µ-Chains at the C-Terminal End

Abstract: Peptide analysis and carboxypeptidase-A digestion have been applied to compare the C-terminal primary structure of µ-chains from nonsecreting and secreting human B cell lines. The expected release of C-terminal tyrosine was seen with internally labeled secreted µ-chains; however, only a small amount of tyrosine was released from internally labeled nonsecreted µ-chains. The greater quantity released of other hydrophobic amino acids Phe, Val, and Leu, together with differences in the kinetics of release suggested that tyrosine was not the C-terminal residue. Since these other amino acids are not found near the C-terminal sequence of secreted µ-chains (-Ser-Asp-Thr-Ala-Gly-Thr-Cys-Tyr-COOH), these results indicate a structural difference between these two forms of µ-chains. Further evidence of a structural difference between these two forms of µ-chains was obtained by the analysis of cyanogen bromide peptides. The C-terminal octapeptide from myeloma and secreted internally labeled µ-chains could be isolated by Sephadex G-50 chromatography and high voltage electrophoresis. In contrast, no such peptide could be obtained from nonsecreted internally labeled µ-chains. These results also strongly indicate that nonsecreted µ-chains (i.e., membrane) possess a different C-terminal structure from those that are secreted. These data have led to the proposal of a hydrophobic C-terminal trailer sequence being responsible for the affinity of nonsecreted µ-chain for the plasma membrane.

Primary structure of murine major histocompatibility complex alloantigens: amino acid sequence studies of the cyanogen bromide fragments of the h-2kb glycoprotein.

Abstract: Radiochemical microtechniques have been used in the amino acid sequence analysis of five major CNBr fragments of the glycoprotein specified by the murine major histocompatibility complex gene H-2kb. These fragments have been tentatively aligned and represent the NH2-terminal 80% of the intact molecule. All amino acids except Asp, Asn, and Gln have been assigned in 128 out of 149 possible positions in the NH2-terminal portions of each of these fragments. These assignments, which represent approximately 50% of the total sequence from these fragments, are listed below in the order of their alignment in the intact H-2Kb molecule: IIIn, -PHSLRYFVTAVSRP(G)L(G)(E)PRYM; IIIa, EVGYV--TEFVRF-S-AE(A)PRYEPR(A)--M; Ib, E-EGPEYWERET-KAK(G)-E-SFR--LRTLL(G)YY--TK; Ia, AALITK-KWE-AGEAERLRAYLEGTC-E-L; Ic, ELVETRPAG-GTF-KWAS-VVPLGKE-YY(T). The unassigned positions represented by dashes in the above sequences may be tentatively assigned as Asp, Asn, or Gln. The NH2-terminal sequence obtained for the H-2Kb molecule was compared to the limited sequence information available for other major histocompatibility complex gene products. An 84% homology (16 of 19 residues) to the H-2Kq and H-2Kk molecules, which are identical to one another in the positions compared, was observed. A similar comparison with 28 of the 31 NH2-terminal residues of HLA-B7 indicated 68% homology. Furthermore, significant homology was observed between H-Kb and HLA-B7 in a region of glycosylation, which occurs between positions 85 and 100 in the two molecules.

Primary structure of the major β-chain of rat haemoglobins

Abstract: The amino acid sequence of the major beta-chain, (II)beta, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the epsilon-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA

Abstract: Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.

The amino-acid sequence of troponin C from frog skeletal muscle.

Abstract: The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103--104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 152 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.