Biomedical subjects

B Dunbar

Publications and source records attributed to B Dunbar.

43 records · Page 3Linked to original sources

PubMed1984

Structure and activity of C1r and C1s.

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.

Amino Acid Sequence↗

PubMed1984

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons.

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N-terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N-terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

Amino Acid Sequence↗

PubMed1984

Human skeletal-muscle aldolase: N-terminal sequence analysis of CNBr- and o-iodosobenzoic acid-cleavage fragments.

Fructose-1,6-bisphosphate aldolase was purified from human skeletal-muscle by affinity elution chromatography. Four CNBr-cleavage fragments were purified by gel filtration, and their N-terminal amino acid sequences were determined. Cleavage with o-iodosobenzoic acid at the three tryptophan residues also yielded fragments suitable for N-terminal sequence analysis. Thus, the sequence of 272 of the 363 residues was established. These sequence results allow many of the discrepancies between the two published rabbit skeletal-muscle aldolase sequences to be resolved. The human aldolase sequence reported here is 96% identical to a "consensus" rabbit aldolase sequence. A comparison with a partial sequence of Drosophila aldolase (103 residues) shows 80% identity. The determination of the amino acid sequence of human aldolase is important for the interpretation of the crystal structure of this enzyme.

Amino Acid Sequence↗

PubMed1983

The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%).

Amino Acid Sequence↗

PubMed1983

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.

Amino Acid Sequence↗

PubMed1983

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase.

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important for the activity of the glycolytic mutase are conserved in the erythrocyte diphosphoglycerate mutase.

Amino Acid Sequence↗

PubMed1982

Primary structure of bovine complement activation fragment C4a, the third anaphylatoxin. Purification and complete amino acid sequence.

Purification of C4a from heat-activated bovine plasma by elution from CM-Sephadex C-50 at pH 7.4 and gel filtration on Sephadex G-50 gives a 20% yield of pure C4a. The complete amino acid sequence of bovine C4a has been determined by automatic sequencer degradation of CNBr and enzymic fragments, and by carboxypeptidase digestion. The 77-residue bovine sequence shows 12 differences from the human sequence with five of these differences occurring in the C-terminal 11 residues. The sequence of C4a confirms earlier suggestions of homology with C3a and C5a: the three sequences show an almost equal number of identities with each other. The six cysteine residues of the 'disulphide knot' are conserved as well as seven other residues including the C-terminal arginine.

Amino Acid Sequence↗