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 mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLYAEMVGYNIL. 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 ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K m of 16.3 μM and k cat of 20.3 s −1 with Z-Arg-Arg-MCA as substrate and a K m of 52.9 μM and k cat of 1.79 s −1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein.