The Disulfide Bridge Sequences of a Serine Protease of Wide Specificity from Streptomyces griseus

The isolation of a Protease from pronase by carboxymethylcellulose and Bio-Rex 70 column chromatography is described. The esterolytic activity of the protease (termed Streptomyces griseus Protease A) towards N-acetyltyrosine ethyl ester was inhibited by diisopropyl fluorophosphate treatment but not by L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone treatment.When oxidized insulin A and B chains were used as substrates in proteolytic specificity studies, it was found that the protease had a wide specificity, with cleavages at peptide bonds containing the carboxyl group of amino acids which did not have electrophilic or polar substitutions on their β-carbon atoms.Interesting features of the amino acid analyses were that the protease molecule contained four half-cystines, three histidines, and one methionine, but no lysines.The two peptic disulfide bridge peptides in the molecule were isolated by G-25 Sephadex chromatography, and paper electrophoresis and chromatography. Sequence analysis of these peptides indicated that one bridge peptide contained the characteristic "active serine" sequence found in Asp–Ser–Gly serine proteases, whilst the other contained the "active histidine" sequence found in these serine proteases.The "active serine" and "active histidine" sequences appeared to be highly homologous with those of the other bacterial and mammalian serine proteases, particularly with those of the α-lytic protease of Myxobacter 495.