Search PubMed⌕ Search

Biomedical subjects

R W Mason

Publications and source records attributed to R W Mason.

At least 55 records · Page 3Linked to original sources
PubMed1989

Synthesis and processing of cathepsin L, an elastase, by human alveolar macrophages.

Cathepsin L was partially purified from lysates of freshly isolated macrophages lavaged from lungs of apparently healthy adults and found to be chromatographically and catalytically identical with liver cathepsin L. Western-blotting analysis showed that lung macrophages contain significant levels of a precursor of cathepsin L (43 kDa) in addition to mature enzyme (25 kDa). After culturing for a further 24 h, the precursor disappeared and a new band, corresponding to 34 kDa, appeared, suggesting that the precursor had been processed to an intermediate form of cathepsin L. Biosynthetic labelling of macrophages in vitro with [35S]methionine followed by immunoprecipitation with the cathepsin L antibody confirmed that the cells synthesize cathepsin L as a 43 kDa precursor that is then processed to the mature form (25 kDa) via a 34 kDa intermediate. The precursor, but not the processed forms, was released into the culture medium. During culture in vitro the 34 kDa intermediate accumulated, and little enzyme was processed to the 24 kDa form, consistent with the immunoblot data. Human lung macrophages contain a 1.5 kb transcript of cathepsin L mRNA, whereas none is detectable in human monocytes. These results establish that differentiation of human macrophages within the lung is accompanied by synthesis and expression of an elastinolytic enzyme, cathepsin L. The altered processing of cathepsin L observed during cultivation in vitro suggests caution in the assessment of the elastinolytic potential of human macrophages based on assay in vitro.

Adult↗
PubMed1989

The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor.

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.

Affinity Labels↗
PubMed1989

Interaction of lysosomal cysteine proteinases with alpha 2-macroglobulin: conclusive evidence for the endopeptidase activities of cathepsins B and H.

The lysosomal cysteine proteinases, cathepsins B, H, and L, were all shown to bind to alpha 2-macroglobulin. The bound enzymes remained active against low-molecular-weight synthetic substrates and bound the active-site-directed inhibitor, benzyloxycarbonyl-125I-Tyr-Ala-diazomethane. Binding of the radiolabeled inhibitor to high-molecular-weight protein on sodium dodecyl sulfate polyacrylamide gels indicated that a proportion of the enzymes was covalently bound to alpha 2-macroglobulin. Cleavage fragments of alpha 2-macroglobulin of Mr 92,000 and 86,000 were seen for cathepsins B, H, and L, indicating cleavage in the bait region. Binding and cleavage were observed for both single-chain and two-chain forms of cathepsin B from human, ox, and pig livers, showing that all active forms of cathepsins B, H, and L are endopeptidases.

Animals↗
PubMed1989

Acute toxicity of combinations of sodium dichromate, sodium arsenate and copper sulphate in the rat.

1. The intraperitoneal treatment of adult male Wistar rats with various combinations of low doses of sodium dichromate (5 mg/kg), sodium arsenate (25 mg/kg) and copper sulphate (5.9 mg/kg) tended to counteract the inherent acute toxicity of each compound. 2. The co-administration of low doses of one or more of the test compounds with a high dose of sodium dichromate (35 mg/kg), sodium arsenate (90 mg/kg) or copper sulphate (23.5 mg/kg) resulted in a significant increase in acute toxicity in comparison with that produced by the administration of high doses of dichromate, arsenate or Cu2+ alone.

Animals↗
PubMed1989

Teratogenicity of combinations of sodium dichromate, sodium arsenate and copper sulphate in the rat.

1. The teratogenicity of sodium dichromate (2 mg Cr/kg), sodium arsenate (5 mg As/kg) and copper sulphate (2 mg Cu/kg) in female Wistar rats was studied following the administration of the test compounds separately and in their various combinations on gestation day 8. 2. The test compounds administered separately and the combination of dichromate plus Cu2+ were non-fetotoxic and either non- or weakly teratogenic, whereas arsenate/Cu2+ was both weakly fetotoxic and teratogenic. 3. Dichromate/arsenate and dichromate/arsenate/Cu2+ caused a marked decrease in mean fetal weight and an increased incidence of fetal resorption and abnormality formation.

Animals↗
PubMed1988

The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B.

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.

Calpain↗
PubMed1988

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

Basement Membrane↗
PubMed1987

The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L.

The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.

Binding Sites↗
PubMed1986

The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L. Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line.

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.

Amino Acid Sequence↗
PubMed1986

Species variants of cathepsin L and their immunological identification.

Cathepsin L variants purified from sheep and ox liver are shown to have similar catalytic properties to those from rat, rabbit and man with regard to activity against the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide and inhibition by benzyloxycarbonyl-Phe-Phe-diazomethane, thus identifying cathepsin L in these species for the first time. All five variants of cathepsin L are shown to be immunologically related by their interaction with antibodies raised to the human enzyme. Sheep liver was found to yield more enzyme than any other species, suggesting that this tissue is a good source of cathepsin L. Cathepsin S, a closely related enzyme, could not be detected in livers of any of these species.

Animals↗