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K J Stevenson

Publications and source records attributed to K J Stevenson.

At least 55 records · Page 3Linked to original sources
PubMed1980

The British Comparative Thromboplastin: the relationship between lipid class composition and procoagulant activity.

Twenty-eight consecutive batches of the reference reagent British Comparative Thromboplastin (BCT) were produced in the National (UK) Reference Laboratory for Anticoagulant Reagents and Control (NRLARC) between 1969 and 1977. The relationship between procoagulant activity and lipid class composition in these batches at various stages of age deterioration on storage has been studied by a modification of the method of high pressure chromatography which allows better definition of the individual lipids. The free fatty acid concentration rose markedly while cconcentrations of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine were reduced. An oxidative process is suggested and supported by the increase in malonaldehyde levels. The method of production of the BCT involves maceration of human brain which causes the breakdown of cell membranes and the release of many potentially oxidative materials from subcellular particles. The loss of procoagulant activity of BCT on storage may be due to the loss of phospholipid or to the increase of inhibitory degradation products, i.e. free fatty acid and malonaldehyde. The examination of the lipid class composition of tissue thromboplastin extracts appears useful, therefore, not only in determining the phospholipids necessary for procoagulant activity, but also in monitoring the deterioration of tissue thromboplastins on storage.

Humans↗
PubMed1980

Procion dyes as affinity ligands and reporter groups for dihydrofolate reductase from Walker 256 carcinoma.

Procion dye - agarose matrices were investigated for isolation of dihydrofolate reductase (FAH2R) from Walker 256 carcinosarcoma. Cibacron blue F3GA, Procion blue MX4GD, Procion blue HERD, and Procion red H3BN covalently bound to agarose adsorbed greater than 85% of pure FAH2R from 100 mM imidazole buffer, pH 6.3, and this enzyme was specifically and quantitatively eluted with 1 mM folate. The capacity and selectivity of the dye-agarose matrices were greater at low dye incorporation. Difference spectroscopy of the FAH2R - Cibacron blue F3GA complex indicated that 2 mol of the dye were bound in hydrophobic environments with each mole of the enzyme. NADPH and folate (at twofold molar excess over enzyme) or 1 M KCl displaced only 1 mol of Cibacron blue F3GA. This dye interacted stoichiometrically in a specific manner with the active site of FAH2R probably spanning the folate and NADP binding sites. The second dye molecule appears to be bound in a nonspecific hydrophobic manner. Selected Procion dye - agarose matrices can be used for partial purification of FAH2R from tumor homogenate.

Animals↗
PubMed1979

Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization.

Lipoamide dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.

Actinomyces↗
PubMed1976

Chymotrypsins from the deer (Cervidae) family. Isolation, partial characterization and primary-structure studies of chymotrypsins A and B from both moose (Alces alces) and elk (Cervus elaphus) pancreas.

1. An anionic and a cationic chymotrypsin (EC 3.4.21.1) were isolated from the pancreas glands of the moose (Alces alces) and elk (Cervus elaphus). The A and B chymotrypsins from each species were purified to homogeneity by (NH4)2SO4 fractionation, affinity chromatography on 4-phenylbutylamine-Sepharose and ion-exchange chromatography on DEAE- and CM-cellulose. 2. The molecular weight and pH optimum of each chymotrypsin were similar to those of the corresponding ox A and B chymotrypsins. 3. The substrate specificities of the chymotrypsins were investigated by digestion of glucagon and the oxidized B chain of insulin. The primary specificity of each chymotrypsin for aromatic amino acid residues was further established by determining the Km and kcat for the hydrolysis of a number of synthetic amino acid ester substrates. 4. The amino acid composition and total number of residues of moose and elk chymotrypsin A were similar to those of ox chymotrypsin A. An even greater similarity was observed among the B chymotrypsins of the three species. 5. The A chymotrypsins of moose and elk were fragmented to their constituent 'A', 'B' and 'C' polypeptide chains by succinylation (3-carboxypropionylation), reduction and alkylation of the native enzymes. In each case, the two major chains ('B' and 'C') were separated and isolated. By comparison of the amino acid compositions of moose, elk and oxy 'B' and 'C' chains, a greater difference was observed among the three A chymotrypsins than was suggested by the amino acid compositions of the native enzymes alone. 6. Peptides were isolated from the disulphide bridge and active-site regions of the A and B chymotrypsins of moose and elk by diagonal peptide-'mapping' techniques. From the amino acid compositions of the isolated peptides (assuming maximum homology) and from a comparison of diagonal peptide 'maps', there was established a high degree of primary-structure identity among the mooae, elk and ox chymotrypsins. Tentative sequences were deduced for the peptides isolated by diagonal peptide 'mapping'. 7. Details of the isolation procedures of the moose and elk chymotrypsins A and B and the amino acid analyses of some peptides obtained by diagonal peptide 'mapping' have been deposited as Supplementary Publication SUP 50064 (27 pages) 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. (1976) 153, 5.

Amino Acid Sequence↗
PubMed1976

Thermomycolin.

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Actinomyces↗
PubMed1975

Characterization of trypsin and elastase from the moose (Alces alces). I. Amino acid composition and specificity towards polypeptides.

Trypsin and elastase isolated from the pancreas of the moose (Alces alces), a member of the Cervidae (deer) family, were characterized with respect to their amino acid composition and specificity towards polypeptides. Moose trypsin possessed 234 residues, based on alanine recoveries equal to 16.0 residues, with a molecular weight calculated at 24 476. Moose trypsin readily hydrolysed peptide bonds in which the carbonyl group was contributed by arginine, lysine and S-2-aminoethylcysteine as indicated by the peptides isolated following hydrolysis of the oxidized and the S-aminoethylated B-chain of insulin. Moose elastase possessed 231 residues, based on alanine recoveries equal to 17.0 residues, with a molecular weight calculated as 24 201. The high lysine (9 residues), low arginine (3 residues) content was in contrast to the opposite situation with porcine elastase and the elastase-like, alpha-lytic protease from Sorangium. The hydrolysis of the oxidized B-chain of insulin by moose elastase was similar to that produced by porcine elastase with major cleavages occurring at Val-12-Glu-13, Ala-14-Leu-15 and Val-18-Cys(O-3H)-19 and minor cleavages occurring at Ser-9-His-10 and Arg-21-Gly-22. The hydrolysis of glucagon with moose elastase produced major cleavages at Thr-7-Ser-8, Ser-11-Lys-12, Val-23-Gln-24 and Leu-26-Met-27. The facile hydrolysis of Arg-17-Arg-18 was also observed and attributed, in part, to trypsin.

Amino Acids↗
PubMed1975

The substrate specificity of thermomycolase, an extracellular serine proteinase from the thermophilic fungus Malbranchea pulchella var. sulfurea.

The specificity of thermomycolase toward glucagon and the oxidized A and B chains of insulin was investigated. Extensive digestion of glucagon occurred when conducted at pH 7.0 and 45 degrees C for 40 min, whereas hydrolysis of only three peptide bonds occurred at pH 7.0 and 28 degrees C for 5 min. A similar situation was observed for the oxidized B chain of insulin, which exhibited only a single major cleavage after 5 min at 25 degrees C. No well-defined specificity for particular amino acid residues was evident, but ready hydrolysis of peptide bonds occurred within sequences containing non-polar residues. This endoproteinase must therefore possess an extended hydrophobic binding site for polypeptides. Thermomycolase hydrolysed acetylalanylalanylalanine methyl ester and elastin-Congo Red at 22 and 8.5 times the rate of porcine elastase respectively. A limited degradation of native collagen and significant hydrolysis of benzyloxycarbonyl-Gly-Pro-Leu-Gly-Pro were suggestive of some collagenase-like activity. No keratinase activity was apparent.

Amino Acids↗
PubMed1974

The selective isolation of an active-site histidine peptide from chymotrypsin-alpha by diagonal peptide 'mapping'. An N-tau-carboxymethylhistidine diagonal peptide "mapping.".

1. The selective isolation of an ;active' histidine peptide from reduced and cyanoethylated chymotrypsin-alpha inhibited with Tos-Phe-CH(2)Cl (l-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained with a His(tauCm) (N(tau)-carboxymethylhistidine) diagonal peptide-;mapping' technique. Performic acid vapours, used between the first and second dimensions of the diagonal peptide ;map', resulted in a peracid rearrangement of the alkylated (Tos-Phe-CH(2))-histidine-57 residue into an N(tau)-carboxymethylhistidine residue. The consequent change in electrophoretic mobility allowed isolation of peptides that contained the ;active' histidine. 2. Peptides containing methionine or S-cyanoethylcysteine were oxidized to their sulphones during the treatment. Peptides in which these residues were N-terminal were selectively isolated on the basis of the change in electrophoretic mobility at pH6.5 which was due to the depression of the pK of the terminal amino group by the inductive effect of the sulphonyl group. 3. An attempt to apply the method to subtilisin BPN' inhibited with l-1-benzyloxycarbonylamido-2-phenylethyl bromomethyl ketone failed to yield a peptide containing N(tau)-carboxymethylhistidine, although peptides containing N-terminal methionine were isolated by the procedure.

Amino Acid Sequence↗
PubMed1973

The isolation and partial characterization of ribonuclease A from Bison bison.

1. Bison ribonuclease was isolated from pancreas glands of Bison bison by acid extraction, (NH(4))(2)SO(4) fractionation, affinity chromatography on Sepharose-5'-(4-aminophenylphosphoryl)uridine 2',3'-phosphate and ion-exchange chromatography on Bio-Rex-70. 2. The selectivity of the affinity column towards bison ribonuclease in heterogeneous protein solutions was greatly improved by employing piperazine buffers at pH5.3, which decreased non-specific interactions of other proteins. Rapid desorption from the affinity column was obtained with sodium phosphate buffer (pH3). 3. Bison ribonuclease has a total amino acid content very similar to ox ribonuclease. Inactivation of bison ribonuclease with iodoacetic acid leads to the formation of 0.62 residues of pi-carboxymethylhistidine and 0.36 residues of tau-carboxymethylhistidine. The amino acid composition of peptides isolated from diagonal peptide ;maps' and also of peptides isolated after pH1.6 and 2.4 two-dimensional high-voltage electrophoresis of a digest of bison ribonuclease labelled with pyridoxal 5-phosphate indicates that there is complete homology between ox and bison ribonucleases. 4. The Schiff-base attachment site of pyridoxal 5-phosphate was identified as lysine-41 by NaBH(4) reduction followed by peptide isolation.

Amino Acid Sequence↗
PubMed1971

Competitive labelling, a method for determining the reactivity of individual groups in proteins. The amino groups of porcine elastase.

1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pK(a) 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pK(a) 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization.

Amino Acids↗