Action of human lysosomal elastase on the oxidized B chain of insulin.
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ABSTRACT:
SUBMITTER: Blow AM
PROVIDER: S-EPMC1164467 | biostudies-other | 1977 Jan
REPOSITORIES: biostudies-other
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Project description:The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.
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Project description:The specificity of cathepsin G, a serine neutral proteinase from human neutrophil leucotyes, was determine dby its action on the insulin B chain. The most susceptible bonds were Phe-24-Phe-25, Leu-15-Tyr-16 and Tyr-16-Leu-17. Other bonds hydrolysed were Leu-6-Cys(O3H)-7, Leu-11-Val-12, Leu-17-Val-18 and Phe-25-Tyr-26. These results suggest that the specificity of cathespin G is closer to that of pig chymotrypsin C than ox Chymotrypsin A. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B2, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161,1.
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Project description:Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.
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Project description:1. The elastase of human spleen was shown to exhibit endopeptidase activity against azo-casein and elastin. 2. Activity against several synthetic substrates was detected, and benzyloxycarbonyl-L-alanine 2-naphthyl ester was found to be a good substrate for routine use. 3. The enzyme showed a broad pH optimum in the range of 8.2-9.2 against azo-casein and the synthetic substrate. 4. The effect of inhibitors on the spleen elastase showed it to be a serine proteinase with a specificity similar to that of porcine pancreatic elastase. 5. Specific antisera were raised against the enzyme, and it was shown to be immunologically identical with the lysosomal elastase of human neutrophil leucocytes.
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