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B W Gibson

Publications and source records attributed to B W Gibson.

84 records · Page 5Linked to original sources
PubMed1985

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

A mass spectrometric assay for novel peptides: application to Xenopus laevis skin secretions.

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fast atom bombardment mass spectrometry (FAB-MS). High performance liquid chromatography (HPLC) of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH and PGLa. Fractions from the HPLC were analyzed directly by FAB-MS to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of FAB-MS, manual Edman degradation, enzymatic digestions and amino acid analyses, identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2,700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by FAB-MS. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from PGLa and the spacer regions in the precursors to xenopsin and caerulein.

Amides↗
PubMed1984

A revised primary structure for neocarzinostatin based on fast atom bombardment and gas chromatographic-mass spectrometry.

The amino acid sequence of the antitumor protein neocarzinostatin was revised on the basis of mass spectrometric studies. Gas chromatographic mass spectrometry on the O-trimethylsilyl polyaminoalcohol derivatives of peptide mixtures derived from tetra S-carboxymethyl-neocarzinostatin were used to partially sequence neocarzinostatin. In addition, fast atom bombardment-mass spectrometric experiments on neocarzinostatin and its tryptic fragments gave the molecular weights of various peptides and, in some cases, partial sequence information. The revised sequence involved reordering of two chymotryptic peptides, the identification of a new di- and tripeptide sequence (Ala-Asp and Ala-Ser-Thr), the repositioning of Trp at position 39, and the assignment of the remaining Asx residues. The revised structure for neocarzinostatin (Mr = 11,105) now shows considerable homology with the other antitumor antibiotic proteins macromomycin and actinoxanthin.

Amino Acid Sequence↗
PubMed1984

Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H.

We report the DNA and primary amino acid sequences of the Streptomyces plicatus enzyme endo-beta-N-acetylglucosaminidase H. Peptide sequence information was derived from enzyme isolated from Streptomyces culture medium using a combination of mass spectrometric methods and conventional techniques, including Edman degradation and carboxypeptidase Y digestion. The DNA sequence was determined by analysis of the Endo-beta-N-acetylglucosaminidase H gene cloned into the Escherichia coli plasmid pBR322 (Robbins, P. W., Wirth , D. F., and Hering , C. (1981) J. Biol. Chem. 256, 10640-10644). The enzyme from Streptomyces medium is 271 (or 269) amino acids in length and has a ragged NH2-terminal sequence beginning primarily with Ala-Pro-Val or Ala-Pro-Ala-Pro-Val. DNA resection experiments as well as the DNA sequence itself suggest that a proenzyme or, more probably, " prepro " enzyme may be the primary product of translation. The long 42 (or 44) residue leader sequence of the preproenzyme shows striking similarities to leader sequences found on proteins secreted by Bacillus species. The leader sequence is partially removed by E. coli and, as reported previously, endo-beta-N-acetylglucosaminidase H made in E. coli appears in both the periplasmic space and in the cell.

Acetylglucosaminidase↗
PubMed1984

Amino acid sequence of human myelin basic protein peptide 45-89 as determined by mass spectrometry.

In order to resolve the uncertainties about the primary structure of human myelin basic protein at residues 45-89, the sequence of this peptide and its tryptic fragments were reinvestigated by fast atom bombardment mass spectrometry. The sequence at positions 77-78 was found to be His-Gly and the sequence at positions 83-84 was shown to be Glu-Asn. The Ser at position 56 was not phosphorylated, whereas the residue at position 46 or 47 showed a heterogeneity of Gly and Ser in this peptide fragment in one of two protein preparations from different patients. These results demonstrate the usefulness of fast atom bombardment mass spectrometry for primary structure information. The corrected sequence of human basic protein peptide 45-89 will permit a more detailed immunochemical analysis of this peptide and its in vivo degradation products.

Amino Acid Sequence↗
PubMed1984

Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences.

Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes. The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically. The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications. This method is very fast and requires little material (5-20 nmol).

Amino Acid Sequence↗
PubMed1983

Primary structures of both subunits of Escherichia coli glycyl-tRNA synthetase.

Escherichia coli glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases which is comprised of two different subunits (in an alpha 2 beta 2 structure). The two coding regions occur in tandem in the order alpha + beta and are synthesized from a single mRNA (Keng, T., Webster, T. A., Sauer, R. T., and Schimmel, P. R. (1982) J. Biol. Chem. 257, 12503-12508). Primary structures of both proteins were determined by DNA sequencing of each coding region and by analysis of tryptic fragments of the enzyme. The alpha-subunit is 303 codons and terminates with TAA; the beta-subunit is 689 codons followed by tandem TAA stops. S1 nuclease mapping of the 3'-end of the two-cistron glyS mRNA showed that it predominantly ends 33/34 bases beyond the tandem stops with an RNA polymerse terminator sequence. Altogether, 43% of the translated polypeptide sequences were confirmed by mass spectrometric analysis of peptide fragments including confirmation of the COOH-terminal end of the beta-chain. This involved determinations, by fast atom bombardment mass spectrometry, of the masses of numerous whole tryptic fragments (with an accuracy of better than 1 Da) and of fragments truncated by one to three cycles of Edman degradations. The primary structures of the two subunits show no homologies with each other and have no internal sequence repeats of significance. While there are no extensive homologies with five other sequenced, or partially sequenced, synthetases, the alpha-subunit has a short sequence which can be aligned with sequences found in functionally important areas of two other synthetases and in uncharacterized parts of a third and fourth synthetase.

Amino Acid Sequence↗
PubMed1983

Primary structure of macromomycin, an antitumor antibiotic protein.

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.

Amino Acid Sequence↗
PubMedDate not supplied

A mass spectrometric method for the identification of novel peptides in Xenopus laevis skin secretions.

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fabms. Hplc of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH, and PGLa. Fractions from the hplc were analyzed directly by fabms to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of fabms, manual Edman degradation, enzymatic digestions, and amino acid analyses identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by fabms. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from spacer regions in the precursors to xenopsin, caerulein, and PGLa.

Amino Acid Sequence↗
PubMedDate not supplied

Lipooligosaccharides: the principal glycolipids of the neisserial outer membrane.

The outer-membrane glycolipids of bacteria that colonize mucosal surfaces that are not routinely bathed by bile acids often lack the long, hydrophilic and neutral polysaccharides that protect the lipid membranes of enteric bacteria from dispersal. The glycolipid from these organisms is properly termed a lipooligosaccharide. A Neisseria strain makes from two to six lipooligosaccharide molecules that range in Mr from 3,150 to 7,100. Different species of Neisseria commonly make lipooligosaccharides of identical Mr and epitope content. Differences in oligosaccharides account for most of the observed physical heterogeneity. Oligosaccharides consist of (1) partially conserved and highly substituted basal oligosaccharides that branch at heptose residues; (2) a linear segment consisting of (hexose)n residues that determines the length of the oligosaccharide; and (3) terminal sequences that are similar to those of glycosphingolipids. Epitope expression is linked to physical heterogeneity and is modified by the molecular environment of the outer membrane. Serotype epitopes are present only on lipooligosaccharides of a certain Mr. Certain lipooligosaccharides regulate complement activation onto the bacterial surface and, hence, immune lysis.

Animals↗