Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization

Miltpain (EC.3.4.22.-) is a cysteine proteinase that preferentially hydroly zes basic proteins, previously found in the milt of chum salmon. Here we re port a similar cysteine proteinase in the milt of the marine Pacific cod. T he enzyme was isolated and purified 6900-fold and with an estimated mass of 63 kDa by gel filtration chromatography and 72 kDa by SDS/PAGE. Cod miltpa in has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and K-m of 11.5 m u M and k(cat) of 19.0 s(-1) with Z-Arg-Arg-MCA. It requires a thiol-induci ng reagent for activation and is inhibited by E-64, iodoacetamide, CA-074, PCMB, NEM, TLCK, TPCK, ZPCK and o-phenanthroline. This proteinase strongly hydrolyzes basic proteins such as salmine, clupeine and histone, and exhibi ts unique substrate specificity toward paired basic residues such as Lys-Ar g, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoe lectric focusing. N-Terminal sequencing gave a sequence of <EVPVEVVRXYVTSAP EK. The cysteine proteinase from Pacific cod very closely matches the previ ously reported miltpain from chum salmon. (C) 2000 Elsevier Science Inc. Al l rights reserved.