MILTPAIN, NEW CYSTEINE PROTEINASE FROM THE MILT OF CHUM SALMON, ONCORHYNCHUS-KETA

A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mas s was estimated as 67,000 by gel filtration column chromatography (Sho dex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoe lectoric point was determined to be 3.9 by isoelectric focusing. The f irst 15 amino acid residues in the N-terminal region were LPSFLY-AEMVG YNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg -Arg MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPC K. The cysteine proteinase exhibited unique substrate specificity towa rd paired basic residues such as Lys-Arg, Arg Arg at the subsites of P 2-P1 and had a K-m of 16.3 mu M and k(cat) of 20.3 s(-1) with 2-Arg-Ar g-MCA as substrate and a K-m of 52.9 mu M and k(cat) of 1.79 s(-1) wit h Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze b asic proteins such as histone, salmine and clupaine but not milk casei n. (C) 1997 Elsevier Science Inc.