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S Doonan

Publications and source records attributed to S Doonan.

At least 91 records · Page 5Linked to original sources
PubMed1976

Large-scale purification and some properties of the mitochondrial aspartate aminotransferase from pig heart.

A method has been developed which allows isolation of 0.3--0.5 g of mitochondrial aspartate aminotransferase in five days starting from 10 pig hearts; the method does not involve initial preparation of mitochondria. Mitochondrial malate dehydrogenase and the cytoplasmic aspartate aminotransferase may conveniently be recovered from side fractions. The product mitochondrial aspartate aminotransferase is homogeneous as judged by various electrophoretic techniques and by N-terminal analysis. Crystals of the enzyme have been obtained both from concentrated, essentially salt-free, solutions and from solutions of ammonium sulphate. The amino acid composition, N and C-terminal amino acid sequences and subunit molecular weight have been determined; these characteristic properties are compared with those of the cytoplasmic isozyme from the same source.

Amino Acid Sequence↗
PubMed1975

Specific enzymic cleavage of polypeptides at cysteine residues.

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the beta chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified cysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide.

Amino Acid Sequence↗
PubMed1975

The primary structure of aspartate aminotransferase from pig heart muscle. Digestion with a proteinase having specificity for lysine residues.

Carboxymethylated aspartate aminotransferase was digested with a proteinase claimed to be specific for lysine residues. Complete cleavage occurred at 12 of the 19 lysine residues in the protein, but at the remaining seven residues cleavage was either restricted or absent. In addition, cleavage was observed at three of the 26 arginine residues. These results are discussed with reference to the amino acid residues adjacent to points of complete or restricted cleavage. The complete primary structure of aspartate aminotransferase, based on these and other studies, is given. Evidence for the assignment of some acid and amide side chains has been deposited as Supplementary Publication SUP 50050 (11 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5. The evidence for the assignment of residue 366 was less conclusive than for the other acid and amide side chains and is, therefore, given in the main paper.

Amino Acid Sequence↗
PubMed1974

Inducibility and some properties of the threonine dehydratase of sheep liver.

The threonine dehydratase extracted from sheep liver is in an essentially inactive form, referred to here as the precursor, but can be activated by incubation at high temperature in alkaline solution. A method for purification of the activated enzyme to a state approaching homogeneity has been devised. The activated enzyme catalyses the deamination of serine but rapidly loses activity during the process. The enzyme inactivated by incubation with serine can be reactivated under the same conditions as those described for the precursor, the kinetic parameters being the same in both cases. Individual sheep livers differ widely in their content of threonine dehydratase, and the possible role of dietary factors in this variability has been examined. The average amount of the enzyme in the livers of sheep fed on high-plant-protein diets did not differ significantly from that in livers obtained from slaughterhouses, but was higher than that in livers from animals fed only on grass or normal concentrates. Large increases in enzyme concentration were, however, obtained when sheep were injected intraperitoneally with a protein hydrolysate. The enzyme from the livers of these sheep could be purified by the same technique and to the same specific activity as the enzyme from the livers of control sheep.

Animals↗
PubMed1973

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase.

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

Amino Acid Sequence↗
PubMed1972

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with pepsin and trypsi trypsin.

Peptides obtained by tryptic digestion of carboxymethylated and maleylated aspartate aminotransferase and of the aminoethylated enzyme were isolated and the complete amino acid sequences of most of them were determined. Digestion of the carboxymethylated protein with pepsin produced a complex mixture of peptides that allowed some overlapping of the tryptic peptides (Fig. 4); in addition, peptides were obtained that had not been found in either of the tryptic digests. From these studies about 400 amino acid residues were identified. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as Supplementary Publication 50011 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.

Alkylation↗
PubMedDate not supplied

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage with pepsin and with Staphylococcus aureus protease.

Results obtained after digestion of mitochondrial aspartate aminotransferase from pig heart with pepsin and with the protease from S. aureus are described. Peptic digestion produced a very complex mixture of peptides, which were purified and analyzed; structural information contained in these peptides covered nearly the entire molecule. Moreover, the lengths of some individual peptides and the peculiar self-overlapping found with families of peptides from adjacent regions were especially useful and interesting. Not all the possible peptides originating after digestion with S. aureus protease were isolated and examined. However, the high specificity of this protease and its usefulness for sequence studies were confirmed. In particular, the S. aureus peptides obtained were important for establishing the amidation state of glutamic acid/glutamine residues.

Amino Acid Sequence↗
PubMedDate not supplied

The primary structure of mitochondrial aspartate aminotransferase from pig heart: peptides obtained by cleavage at basic residues.

Results obtained as part of a study of the primary structure of mitochondrial aspartate aminotransferase from pig heart are described. In particular, the S-aminoethylated protein was digested with trypsin and with the lysine specific protease from A. mellea. In the first case peptides contained 221 out of the total of 401 amino acid residues in the protein were obtained. By contrast the digest with A. mellea protease was not examined exhaustively and six peptides containing 49 amino acid residues were isolated. Digestion of the trifluoroacetylated and S-aminoethylated protein with A. mellea protease yielded a mixture of large fragments three of which, containing 89 amino acid residues, are described here. The combined results of these three digests yielded 66.6% of the total structure, concentrated mainly in the N-terminal half of the protein.

Amino Acid Sequence↗