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R J Beynon

Publications and source records attributed to R J Beynon.

At least 55 records · Page 3Linked to original sources
PubMed1992

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.

Amino Acid Sequence↗
PubMed1991

The astacin family of metalloendopeptidases.

Molecular cloning of a human intestinal brush border metalloendopeptidase (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, PPH) and a mouse kidney brush border metalloendopeptidase (meprin A) has revealed 82% identity in the NH2-terminal amino acid sequences (198 residues) of the mature enzymes. Furthermore, searching of protein sequence data bases with the inferred peptide sequences as probes revealed strong similarities to astacin, a crayfish digestive protease, and an NH2-terminal domain of a human bone morphogenetic protein (BMP-1). Meprin A and PPH both have approximately 30% identity with astacin and BMP-1. Multiple alignment analysis indicated that 37 residues, including 3 cysteine residues, are strictly conserved for the four proteins in a sequence frame equivalent to the complete 200-amino acid astacin sequence. The four proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases, within an extended sequence of HEXXHXXGFXHE which is unique to this subgroup of metalloendopeptidases. In addition, the four proteins have 54% identity in a 24-amino acid sequence that includes the putative active site. A fifth protein, Xenopus laevis developmentally regulated protein UVS.2, also shares sequence identity with the metalloendopeptidases. These data provide strong evidence for an evolutionary relationship of these proteins. It is suggested that this new family of metalloendopeptidases be called the "astacin family."

Amino Acid Sequence↗
PubMed1991

The expression of glycogen phosphorylase in normal and dystrophic muscle.

Specific cofactor labelling was employed to determine the degradation rate of glycogen phosphorylase in normal adult C57BL/6J mice and their dystrophic counterparts (C57BL/6Jdy/dy). The rate constant for the decay of phosphorylase-bound label was 0.125 day-1 in normal muscle and 0.49 day-1 in dystrophic muscle, i.e. a lower rate of catabolism of phosphorylase in dystrophic muscle. Quantitative Northern-blot analyses of total RNA isolated from normal and dystrophic muscle indicated that the abundance of phosphorylase mRNA as a percentage of total RNA was approx. 40% lower in dystrophic muscle. The specific activity of phosphorylase in dystrophic muscle is approx. 60% lower than in normal muscle, and is elicited by a lower rate of turnover of the enzyme, i.e. both synthesis and degradation are decreased.

Animals↗
PubMed1991

Immunological characterisation of different meprin species in mice.

Meprin-a is a metalloendopeptidase present at high levels in the kidney brush border of some inbred mouse strains. Meprin-b is a latent metallo-endopeptidase, activated by trypsin-mediated proteolysis in vitro, that is present at similar activities (after activation) in all mouse strains. Meprin (a mixture of a and b forms) was purified from a high-meprin Mep-1a/a animal, and Lys-C peptides of this preparation were sequenced. The sequence data were used to direct the synthesis of peptides that were conjugated to albumin and used as immunogens. One of these antisera was specific to meprin-b and thus provided a specific tool to monitor expression of this form of meprin in different mouse strains.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of thaumatopain, a cysteine protease from the arils of the plant Thaumatococcus daniellii.

Aqueous extracts of the aril of the seed of Thaumatococcus daniellii contain, in addition to the intensely sweet protein thaumatin, a cysteine protease that we have termed thaumatopain. Thaumatopain has been purified by ion-exchange chromatography from arils, and is a monomeric protein of Mr 30,000. The protease strongly resembles papain in proteolytic activity, pH optima, susceptibility to inhibitors of cysteine proteases and in N-terminal sequence. The protease has also been identified in crude aril extracts by affinity labelling with iodo[14C]acetate. Thaumatopain is responsible for the cysteine protease activity previously attributed to thaumatin. Thaumatin is digested by thaumatopain at neutral to alkaline pH values.

Amino Acid Sequence↗
PubMed1991

Genetic differences in turnover of glycogen phosphorylase in broiler and layer chickens.

Specific labelling of glycogen phosphorylase with the precursor of the cofactor has allowed definition of the turnover parameters of the enzyme in the pectoralis of rapidly growing broiler chickens, and slowly growing layer chickens. Selection for rapid growth rate in broilers has resulted in a lower rate of turnover of phosphorylase, but the difference between rates of synthesis and degradation is maintained, which contrasts markedly with the age-dependent coalescence of the two values in layer chickens.

Age Factors↗
PubMed1991

Metalloendopeptidase activity in urine of rodents.

The brush border membrane of mice and rats contains a phosphoramidon-insensitive metalloproteinase, meprin (neutral endopeptidase-2; NEP-2). The role of meprin is unknown, but we have shown that urine from these species contains insulin B chain degrading activity that is due to a phosphoramidon-insensitive metalloendopeptidase. By enzymic and immunological criteria, it is likely that this activity is due to meprin, and introduces the possibility that this enzyme may have a role in urinary function.

Animals↗
PubMed1990

Effect of denervation on the expression of glycogen phosphorylase in mouse skeletal muscle.

After sciatectomy of the left hind-limb of C57BL/J mice, a denervation-induced muscular atrophy ensued and was accompanied by a decrease in the specific activity of glycogen phosphorylase to approx. 25% of control values. The cofactor of phosphorylase, pyridoxal 5'-phosphate, was used as a specific label in the determination of the degradation rate of the enzyme following nerve section. After a delay of 3-4 days, phosphorylase was degraded approx, twice as rapidly in the denervated gastrocnemius (0.20 day-1) as in the control muscle (0.12 day-1). The effect of denervation on phosphorylase mRNA was measured by quantitative Northern-blot analysis using a rat skeletal-muscle phosphorylase cDNA probe. After an initial rapid decline, phosphorylase mRNA levels stabilized in denervated muscle at 50% of the value measured in the contralateral control muscle.

Animals↗
PubMed1990

Molecular heterogeneity in McArdle's disease.

Biopsies were taken from a group of eleven patients with McArdle's disease, a congenital deficiency in muscle glycogen phosphorylase. The biopsies were screened by Western and Northern blotting for phosphorylase protein, phosphorylase-bound pyridoxal-5'-phosphate (the cofactor of the enzyme) and for phosphorylase mRNA. Of the eleven patients, three expressed phosphorylase mRNA at near normal levels and at the expected size. One of these patients also expressed low levels of phosphorylase protein that correlated with a small amount of measurable phosphorylase activity. These data support the contention of molecular heterogeneity in the presentation of this phenotype.

Antibodies, Monoclonal↗
PubMed1990

Proteolytic activity in mouse urine: relationship to the kidney metallo-endopeptidase, meprin.

Meprin, a brush border kidney metallo-endopeptidase is present as the major endopeptidase in mouse urine. The enzyme is freely soluble and can be detected enzymically or immunologically. Mice can be partitioned into two phenotypes that differ by 10-20-fold in the amount of meprin in kidney membranes; this phenotypic variation is reflected in urinary activities. We propose a role for meprin in the degradation of other urinary proteins.

Animals↗
PubMed1990

3,4-Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b.

3,4-Dichloroisocoumarin (3,4-DCI) is a highly reactive, mechanism-based inhibitor of serine proteases. We show here that glycogen phosphorylase b is also inactivated by this inhibitor, in a mechanism that parallels the inactivation of serine proteases, but involving multiple sites of covalent modification. Such a process may compromise studies in which 3,4-DCI is used to arrest proteolysis of a second native protein which may itself be modified.

Animals↗
PubMed1990

A cryptic meprin-like proteolytic activity in mouse kidney brush border membranes.

1. Inbred mouse strains differ markedly in the expression of a kidney brush border metalloendopeptidase, meprin-a. 2. Brush border preparations from mice of the low-meprin-a phenotype (specific activities less than 5% of the high-meprin-a trait) contain a metallo-endopeptidase, meprin-b, that is larger than meprin-a, and which is inactive unless the membrane preparations are treated with trypsin. 3. This cryptic metallo-endopeptidase has been previously postulated to be a stalled precursor of meprin-a. 4. We show here that meprin-b is present in all mice-high and low meprin-a phenotypes--and that this activity is similar in substrate specificity and amount present in the brush border. 5. Meprin-b may therefore be a distinct gene product that is independent of meprin-a phenotype.

Animals↗