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Biomedical subjects

R J Beynon

Publications and source records attributed to R J Beynon.

107 records · Page 6Linked to original sources
PubMed1983

Certain mouse strains are deficient in a kidney brush-border metallo-endopeptidase activity.

Preparations of microvilli from kidneys of BALB/c mice contain an alkaline metallo-endopeptidase, meprin (metallo-endopeptidase from renal tissue). Certain genealogically related inbred mice are markedly deficient in meprin activity. The meprin-deficient strains (CBA/J and C3H/HeJ) exhibit normal levels of other brush-border enzymes: alkaline phosphatase, aminopeptidase M and another proteinase, a phosphoramidon-sensitive neutral endopeptidase. Meprin deficiency cannot be attributed to a shift in pH optimum and is unlikely to be due to the presence of endogenous inhibitors.

Animals↗
PubMed1983

Tissue and subcellular distribution of enzymes inactivating leupeptin.

Leupeptin, a proteinase inhibitor obtained from culture filtrates of Streptomyces sp., has been proposed as a valuable agent for the restriction of proteolysis in vivo. We have previously shown that mammalian tissues possess an enzymic system that is capable of inactivating leupeptin (Beynon RJ, Brown CP & Butler PE, 1981, Acta Biol. Med. Germ. 40, 1539-1546). This paper demonstrates the ubiquitous distribution of the inactivating system throughout mammalian tissues and provides evidence for the location of the enzyme(s) in the soluble fraction of the cell.

Animals↗
PubMed1982

The chymotrypsin-catalysed activation of bovine liver glutamate dehydrogenase.

1. Ox liver glutamate dehydrogenase is activated by bovine pancreatic alpha-chymotrypsin, but the extent of activation is dependent on the age of the dehydrogenase preparation. 2. The degree of activation is constant and the pseudo-first-order rate constant of activation is directly proportional to the concentration of proteinase used. 3. Commercial preparations of alpha-chymotrypsin differ in their ability to produce a secondary inactivation phase, and this was shown to be due to low tryptic contamination. The 'superactive' form of glutamate dehydrogenase has an increased sensitivity to tryptic inactivation as compared with the native enzyme. 4. Analysis of the activation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the subunit molecular weight of 'superactive' glutamate dehydrogenase differs by less than 5% from that of the native subunit.

Animals↗
PubMed1982

A microcomputer program for analysis of nucleic acid hybridization data.

The study of nucleic acid hybridization is facilitated by computer mediated fitting of theoretical models to experimental data. This paper describes a non-linear curve fitting program, using the 'Patternsearch' algorithm, written in BASIC for the Apple II microcomputer. The advantages and disadvantages of using a microcomputer for local data processing are discussed.

Computers↗
PubMed1982

The effect of ionic environment on pig heart mitochondrial malate dehydrogenase.

1. The effect of ionic environment on pig heart mitochondrial malate dehydrogenase was investigated by means of two low resolution conformational probes, thermal stability and proteolytic liability. 2. High ionic strength and high enzyme concentrations stabilize the enzyme towards thermal inactivation, probably by maintaining the enzyme in the dimeric form. 3. Proteolysis of malate dehydrogenase under conditions where the enzyme is thermally stable indicates a secondary ionic effect on structure. 4. Attempts to demonstrate dissociation of native malate dehydrogenase dimer into its constituent subunits using gel filtration on Sephacryl S-200 proved inconclusive. At low ionic strength malate dehydrogenase does not exhibit normal gel filtration behaviour.

Animals↗
PubMed1981

Purification and characterization of a metallo-endoproteinase from mouse kidney.

A metallo-endoproteinase was purified from mouse kidney. The enzyme was solubilized from the 100 000 g sediment of kidney homogenates with toluene and trypsin, and further purified by fractionation with (NH4)2SO4. DEAE-cellulose chromatography and gel filtration. The molecular weight of the metalloproteinase was estimated by gel filtration on Sepharose 6B to be 270 000--320 000. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of 2-mercaptoethanol, a single major protein with a mol.wt. of 81 000 was observed. Thus the active enzyme is an oligomer, probably a tetramer. It is a glycoprotein and has an apparent isoelectric point of 4.3. Kidney homogenates and purified preparations of the metalloproteinase degraded azocasein optimally at pH 9.5 and at I 0.15--0.2. The activity was not affected by inhibitors of serine proteinases (di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride), cysteine proteinases (4-hydroxymercuribenzoate, iodoacetate), aspartic proteinases (pepstatin) or several other proteinase inhibitors from actinomycetes (leupeptin, antipain and phosphoramidon). Inhibition of the enzyme was observed with metal chelators (EDTA, EGTA, 1,10-phenanthroline), and thiol compounds (cysteine, glutathione, dithioerythritol, 2-mercaptoethanol). The metalloproteinase degraded azocasein, azocoll, casein, haemoglobulin and aldolase, but showed little or no activity against the synthetic substrates benzoylarginine 2-naphthylamide, benzoylglycylarginine, benzyloxycarbonylglutamyltyrosine or acetylphenylalanyl 2-naphthyl ester. This metalloproteinase from mouse kidney appears to be distinct from previously described kidney proteinases.

Animals↗
PubMed1981

Alkaline proteolytic activity from smooth muscle of mouse small intestine.

An alkaline proteolytic activity from the smooth muscle of mouse small intestine has been separated and characterised. The activity sedimented after high-speed centrifugation, but was released into the soluble phase after treatment with 2.0 M KCl. The proteinase was found to be sensitive to salt concentration and the activity was maximal between 0.1-0.5 M NaCl/KCl and pH 9.5. This activity was completely inhibited by di-isopropylphosphoro fluoridate suggesting that it is a serine endopeptidase. The proteinase was identified as chymotrypsin-like due to the inhibition observed with the agents chymostatin, lima bean and soya bean trypsin inhibitor. These characteristics of the alkaline proteinase resemble the properties of the mast cell enzyme, chymase. The enzyme activity was measured in 48/80 treated animals and the mutant strain w/wv, which do not contain mast cells. No significant reduction in the enzyme activity was observed.

Animals↗
PubMed1981

The inactivation of streptomyces-derived proteinase inhibitors by mammalian tissue preparations.

The proteinase inhibitors leupeptin and chymostatin are inactivated by preparations of perfused mouse liver. The inhibitors are degraded maximally at neutral or alkaline pH values. The inactivating enzymes are inhibited by Dip-F and pms-F but not by EDTA, 1,10 phenanthroline or iodoacetic acid. The significance of these results is discussed in terms of the potential of the inhibitors as antiproteolytic drugs.

Animals↗
PubMed1978

The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin.

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With (32)P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.

Adenosine Monophosphate↗
PubMed1978

The inactivation of native enzymes by a neutral proteinase from rat intestinal muscle.

1. The solubilization and partial purification of a proteinase from the intestinal smooth muscle of rats fed on protein-free diets are described. 2. It has a mol.wt. of about 33000 and it is stable over a narrow pH range. 3. From its susceptibility to known modifers of proteolytic enzymes, it appears to be a serine proteinase of a trypsin-like nature. Active-site titration with soya-bean trypsin inhibitor shows that the concentration of proteinase was about 3 microgram/g wet wt. of intestinal smooth muscle. However, the muscle proteinase demonstrates a marked ability for inactivating enzymes in their native conformation at neutral pH. It is about 100 times more efficient than pancreatic trypsin when the inactivating activities are compared on an approximately equimolar basis. 4. Inactivation of the substrate enzymes is accompanied by limited proteolysis, as demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 5. An endogenous inhibitor was separated from the proteinase by fractionation with (NH4)2SO4. 6. Contamination of the muscle tissue by lumen, mucosal or blood proteinases and inhibitors is shown to be unlikely. 7. A role for the neutral trypsin-like proteinase in initiating the degradation of intracellular enzymes is considered.

Animals↗
PubMed1977

A neutral protease from rat intestinal muscle. A possible role in the degradation of native enzymes.

A membrane-limited protease has been solubilised and partially purified from the intestinal smooth muscle of rats fed on protein free diets. This neutral protease has a mol. wt. of around 33,000 and from its susceptibility to several known modifiers of proteolytic enzymes, it appears to be trypsin-like. It is stable over a relatively narrow pH range and it appears to have a markedly enhanced ability over trypsin for inactivating substrate enzymes in their native conformations through limited proteolysis. The rate of inactivation of substrate enzymes can be modulated by cofactors, allosteric ligands, or by changes in ionic strength. In addition, a specific protein inhibitor of the protease has been measured and levels of this are high in animals fed on normal diets. On administration of protein free diets, the inhibitory activity is depleted. Contamination of the muscle tissue by lumenal, mucosal or blood proteases and inhibitors has been excluded. A role for the neutral protease in initiating the turnover of intracellular enzymes is postulated.

Animals↗
PubMed1977

The susceptibility of glycogen phosphorylase to inactivation by endogenous and exogenous proteases.

Phosphorylases a and b were inactivated very rapidly by a neutral, trypsin-like protease from rat intestinal muscle. With 32P-phosphorylase a as substrate, it was shown that the initial event in the inactivation was the release of a small, phosphopeptide from the N-terminus of the enzyme, leaving the original 100,000 subunit form virtually unchanged. Subsequent proteolysis was very limited, producing 85, 70 and 65,000 mol. wt. derivatives. The effects of several allosteric modulators of phosphorylase on the rates of inactivation of the two enzymes were studied. Removal of the pyridoxal phosphate cofactor from phosphorylase increased the susceptibility of the b form by three fold while the a form was unaffected. By comparison of these effects with those obtained from digestion with trypsin and chymotrypsin, it is concluded that the intestinal muscle protease has a markedly enhanced ability for inactivating enzymes in their native conformation. Assuming that this property is reflected in vivo, a possible role such neutral proteases in initiating protein degradation is advanced.

Animals↗
PubMedDate not supplied

The greater susceptibility of North Ronaldsay sheep compared with Cambridge sheep to copper-induced oxidative stress, mitochondrial damage and hepatic stellate cell activation.

Sheep of the semi-feral North Ronaldsay (copper-sensitive) and domesticated Cambridge (copper-tolerant) breeds were compared in respect of pathological changes and protein expression in the liver as a result of excessive dietary copper. Acute mitochondrial damage and hepatic stellate cell (HSC) activation with collagen synthesis occurred in response to moderate copper overload in North Ronaldsay but not in Cambridge sheep. Mitochondrial degradative changes occurred either as ballooning degeneration and rupture with subsequent autophagic degradation or as mitochondrial matrical condensation (pyknosis). In North Ronaldsay sheep prolonged exposure to copper produced mitochondrial hyperplasia and hypertrophy, and nuclear damage with necrosis. Cytosolic isocitrate dehydrogenase (IDH), an enzyme responsive to oxidative stress, was induced in the liver of Cambridge sheep receiving a Cu-supplemented diet but was undetectable in the non-supplemented control sheep. Conversely, IDH was detected at similar levels in both control and copper-supplemented North Ronaldsay sheep, indicating a lower threshold response, and an enhanced susceptibility, to oxidative stress. "Upregulation" of mitochondrial thioredoxin-dependent peroxidase reductase (antioxidant protein-1) in the hepatic cytosol of the North Ronaldsay (but not Cambridge) sheep affirmed the increased susceptibility of the mitochondria to Cu-induced oxidative stress in this breed. Likewise the upregulation of cathepsin-D indicated increased lysosomal activity and HSC activation. The findings may be relevant to copper toxicosis in human infants.

Animals↗