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R J Beynon

Publications and source records attributed to R J Beynon.

At least 73 records · Page 4Linked to original sources
PubMed1989

Further evaluation of cofactor as a turnover label for glycogen phosphorylase.

1. The cofactor of glycogen phosphorylase, pyridoxal phosphate (PLP), is stably associated with the enzyme and has been used as a label in the determination of the turnover of the skeletal muscle enzyme in vivo. 2. Mice were injected with radiolabelled pyridoxine that was subsequently converted to PLP and incorporated into phosphorylase. 3. In this study we have resolved phosphorylase-bound label from that associated with the other PLP-containing enzymes and free label by affinity and size-exclusion chromatography. 4. The decay of radioactive pools was assessed after an extended period post-injection to minimize the effects of isotope reutilization. 5. These modifications have allowed refinement of our previous estimate of the rate of degradation of muscle phosphorylase.

Animals↗
PubMed1989

Regulation of a membrane-bound proteinase in mammalian cells.

Cellular proteolytic enzymes are regulated by multiple mechanisms that affect gene expression, enzyme concentration, and enzyme activity. The expression of the membrane-bound metallo-endopeptidase, meprin, in different mammalian species, strains, tissues and cell types is highly variable. Meprin is exclusively localized to the plasma membrane, and this determines the types of substrates the enzyme will encounter and the microenvironment for activity. Recent studies have revealed an inactive form of a meprin-like proteinase that can be activated in vitro by proteases, and this raises the possibility of regulation of enzyme activity at the cell surface in response to environmental stimuli. In this chapter, the current knowledge about meprin, and the meprin-like inactive forms in mouse kidney is discussed.

Animals↗
PubMed1988

A Macintosh Hypercard stack for calculation of thermodynamically-corrected buffer recipes.

This paper describes a stack for the Apple Macintosh Hypercard environment that facilitates the calculation of a set of thermodynamically-corrected pH buffers. Presented in the familiar Macintosh mouse-based environment, the program allows comprehensive buffer design and gives the user full control of buffer species, temperature, ionic strength, pH and choice of counter-ion to maintain the ionic strength at a fixed value. Addition of new buffers to the stack is straightforward and the recipes that are generated, including the weights of the various components, can be displayed on the screen or saved to a disk file for subsequent printing.

Algorithms↗
PubMed1987

The effect of analogues of chymostatin on lysosomal and non-lysosomal components of protein degradation in isolated hepatocytes.

Of the proteinase inhibitors derived from Streptomyces spp., chymostatin is the most effective inhibitor of non-lysosomal proteolysis. As part of a systematic study of the structural features of the chymostatin molecule that are responsible for this inhibitory activity, a series of fifteen di- and tripeptide analogues of chymostatin were tested for their ability to suppress protein degradation in isolated primary hepatocytes. Protein degradation was assessed in two ways: by the release of radiolabel from proteins prelabelled in vivo (to which both lysosomal and non-lysosomal processes contribute) and by the rate of inactivation of tyrosine aminotransferase, a process that is exclusively non-lysosomal. All inhibitors were relatively non-toxic and did not affect the intracellular ATP levels, although some suppression of gluconeogenesis was observed in the presence of leupeptin, chymostatin or the analogues. Tripeptide phenylalanine aldehydes or semicarbazones were at least as effective as chymostatin in reducing protein degradation, whereas peptide alcohols were relatively ineffective. Replacement of the basic capreomycidine moiety in chymostatin with an arginine residue improved the inhibitory activity but equally, substitution of the arginine residue with an uncharged norleucine residue was without significant effect. The structural features that are optimal for inhibition of chymotrypsin or other serine proteinases (previously defined) are not as critical for inhibition of protein degradation in vivo.

Adenosine Triphosphate↗
PubMed1987

Degradation artefacts during sample preparation for sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Preparation of samples for sodium dodecyl sulphate polyacrylamide gel electrophoresis routinely involves heating the protein in solution containing detergent and reducing agent for at least two minutes. Here we show that this treatment causes fragmentation of the protein glycogen phosphorylase, whether purified or as a component of a skeletal muscle preparation. The fragments are detected as minor bands on western blots and represent the products of discrete breakage point in the peptide sequence. Protease inhibitors cannot suppress the fragmentation. Such small amounts of immunoreactive fragments may be incorrectly identified on western blots as contaminants that were originally present in the antigen preparation. They may also be a source of ambiguity in studies that search for degradation intermediates during proteolysis.

Animals↗
PubMed1986

Effect of glycerol on thermolysin-catalyzed peptide bond synthesis.

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl-L-aspartic acid with L-phenylalanine methyl ester to form N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z X Asp X Phe X OMe), the precursor to the sweet-tasting compound L-aspartyl-L-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl-L-glycyl-L-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z X Asp X Phe X OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.

Catalysis↗
PubMed1986

Preparation and purification of the proteinase inhibitor, leupeptin, from culture filtrates of Streptomyces lavendulae.

Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) was produced as an extracellular metabolite by Streptomyces lavendulae. Addition of lysine to the growth medium stimulated leupeptin production from 200 to 1400 micrograms/ml. Incubation of late exponential phase mycelium in a synthetic medium was used to prepare single labelled (3H) and double labelled (3H/14C) inhibitor for metabolic studies. An improved purification scheme that generates leupeptin of high purity with good recoveries is also reported.

Carbon Radioisotopes↗
PubMed1986

Catabolism of intracellular protein: molecular aspects.

All living cells regulate the content and composition of their resident proteins, but the mechanisms by which this is accomplished are not understood. The process of protein degradation has an important role in determining steady state and fluctuations of protein concentrations in mammalian cells. This process may be regulated by innate properties of the protein substrates, by factors that interact or "brand" proteins for degradation or by the degradative machinery of the cell. For a specific protein, there appears to be a committed step, an irreversible event that leads to rapid and extensive degradation. That initial event may or may not involve 1) proteolysis, 2) a nonproteolytic covalent modification or branding event (e.g., oxidation, ubiquitin conjugation), 3) denaturation or unfolding of the protein, or 4) sequestration. The degradative machinery of cells may either recognize proteins committed to degradation or initiate degradation, but the process must be selective because there is great heterogeneity in the rates of degradation for different proteins of one cell. The degradative process certainly requires proteases, and it is probable that lysosomal and extralysosomal proteases are involved in the catabolism of cellular proteins. We review here briefly what is currently known about the factors that may determine the half-life of a protein in a mammalian cell, the role of the protein substrate and sequestration in the process, the proteolytic and nonproteolytic enzymes that may initiate the degradative process, and the regulation of extensive degradation of proteins in cells.

ATP-Dependent Proteases↗
PubMed1986

Metalloendopeptidases of the mouse kidney brush border: meprin and endopeptidase-24.11.

Two metalloendopeptidases, meprin and endopeptidase-24.11 ("24.11"), were isolated from mouse kidney membranes, and their structural and catalytic properties were investigated. The enzymes both cross-react with antibodies prepared in rabbits against purified preparations of meprin; thus they share some immunologic determinants. Meprin and 24.11 have similar subunit molecular weights of 85 000 and 90 000, respectively, as demonstrated after sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. However, under non-reducing conditions, meprin migrates as an oligomer while 24.11 remains monomeric. This and other data indicate that meprin subunits are linked by disulfide bridges, whereas endopeptidase-24.11 subunits are not covalently linked. Both endopeptidases hydrolyze insulin B chain and are totally inhibited by EDTA and o-phenanthroline. The activity of 24.11 against insulin B chain was totally inhibited by low concentrations of phosphoramidon (less than 2 nM), whereas meprin was not inhibited by concentrations of this inhibitor as high as 20 microM. Large proteins are not substrates for endopeptidase-24.11, while meprin degrades proteins such as azocasein rapidly (apparent Km = 0.65 mg/ml). Meprin appears to require an extended polypeptide chain in substrates while 24.11 prefers smaller peptides as substrates. Both endopeptidases have a preference for peptide bonds that contain hydrophobic amino acids. With the octapeptide angiotensin II as substrate, both enzymes hydrolyze the central Tyr-Ile bond; 24.11 also cleaves at Arg-Val and Ile-His. The two endopeptidases show many similarities immunologically, structurally and catalytically, however, they display distinct characteristics which may be physiologically important.

Angiotensin II↗
PubMed1986

Turnover of skeletal muscle glycogen phosphorylase.

Glycogen phosphorylase is a major sarcoplasmic protein and is one of the key regulatory enzymes in the control of glycogen utilisation. In C57BL/6J mice, the activity of the enzyme decreases under muscle-wasting conditions, including denervation-induced atrophy and muscular dystrophy. The cofactor of this enzyme, pyridoxal phosphate, has been employed as a specific label to permit measurement of the rate of degradation of the enzyme in vivo. In both of the muscle-wasting conditions, the decay of protein-bound label occurs at a higher rate than in normal animals, suggesting accelerated degradation of the enzyme. Additionally, we have used a monoclonal antibody, specific for pyridoxal phosphate, as a sensitive probe to search for degradation intermediates of this enzyme in vivo.

Animals↗
PubMed1985

The turnover of skeletal muscle glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label.

The turnover of glycogen phosphorylase has been measured using the cofactor, pyridoxal phosphate, as a label specific for this enzyme in skeletal muscle. Radiolabelled pyridoxine administered in vivo is incorporated into a protein-bound fraction in skeletal muscle, shown by several criteria to be equivalent to glycogen phosphorylase. This pool of radiolabel disappears slowly with a half-life of 11.9 days, taken to be a good estimate of the intracellular half-life of the enzyme. The use of the cofactor in this fashion minimises overestimation of half-life that results from reincorporation of the label. Further, premature dissociation of the cofactor from native enzyme, which would lead to underestimation of half-life, is unlikely. At the level of sensitivity given by this method there was little evidence for the appearance of pyridoxal phosphate-labelled degradation intermediates of the enzyme.

Animals↗
PubMed1985

Deficiency of a mouse kidney metalloendopeptidase activity: immunological demonstration of an altered gene product.

Meprin, an 85,000 molecular weight metalloendopeptidase is a major component of the kidney brush border membrane in mice. Some inbred mouse strains exhibit low levels of meprin activity. These strains were characterized by little, if any, protein in brush border preparations corresponding to the native enzyme. However, material exhibiting partial identity to meprin was identified by Ouchterlony immunodiffusion. Immunoblots of brush border proteins confirmed that this immunoreactive material was present but of higher molecular weight than the native enzyme. The implication of these data is that the structural gene for meprin is expressed, albeit incorrectly, in the low-meprin strains.

Amino Acids, Sulfur↗
PubMed1985

The effect of synthetic analogues of chymostatin upon protein degradation in isolated skeletal muscle.

A series of peptides based on the structure of the proteinase inhibitor chymostatin were tested for their toxicity and ability to suppress protein degradation in the isolated mouse diaphragm. The inhibitory activities of the analogues were very similar, in marked contrast to their disparate abilities as inhibitors of chymotrypsin. Toxicity was determined by measurement of the rates of protein synthesis and of leakage of lactate dehydrogenase into the incubation medium. No significant toxicity was measurable at concentrations of inhibitor that were effective at suppressing proteolysis. The structural features of the chymostatin molecule may be less than optimal for suppression of proteolysis in muscle.

Animals↗