RESUMO
Pure extracellular serine protease was isolated from the culture filtrate of Halobacterium halobium by bacitracin-Sepharose affinity chromatography. The enzyme activity was completely and irreversibly lost if the NaCl concentration fell below 2 M. The protease consists of one polypeptide chain with a molecular weight of 41,000. It is characteristically enriched in Asx and Glx content, whereas the level of basic amino acids in the enzyme molecule is unusually low. The protease shows a preference for leucine in the carboxylic side of the scissile bond of the substrate, cleaving the B-chain of oxidized bovine insulin only at the Leu15-Tyr16 bond and liberating p-nitroaniline from L-pyroglutamyl-L-alanyl-L-alanyl-L-leucine-p-nitroanilide.
Assuntos
Endopeptidases/análise , Halobacterium/enzimologia , Aminoácidos/análise , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Peso Molecular , Serina Endopeptidases , Cloreto de Sódio/farmacologiaAssuntos
Líquidos Corporais/enzimologia , Líquido Intracelular/enzimologia , Subtilisinas/metabolismo , Aminoácidos/análise , Anilidas , Animais , Bacillus/enzimologia , Bovinos , Cromatografia em Gel , Endonucleases/metabolismo , Insulina/metabolismo , Oligopeptídeos , Coelhos , Ribonuclease Pancreático , Ribonucleases/metabolismo , Especificidade por SubstratoRESUMO
It was demonstrated that crystals of entomopathogenic protein from Bac. thuringiensis contain admixture of proteinase either adhered to their surface on inconponated into crystal lattice defects. A proteolytic action, particularly when enhanced by crystal dissolution, causes progressive degradation of crystal proteins with molecular weights of 140 000--129 000 down to the components with smaller molecular weights. This may, at least, partially account for the contradictions in the literature data on crystal composition. Using synthetic peptide substrates and specific inhibitors, it was shown that the enzymes incorporated into crystals belong to serine and metalloproteases. The presence of leucine aminopeptidase was also noted. A method for enzyme separation from crystal has been developed.