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 hydrolyzes basic proteins, previously found in the milt of chum salmon. Here we report a similar cysteine proteinase in the milt of the marine Pacific cod. The 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 miltpain has an optimum pH of 6.0 for Z-Arg-Arg-MCA hydrolysis, and Km of 11.5 microM and kcat of 19.0 s-1 with Z-Arg-Arg-MCA. It requires a thiol-inducing 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 exhibits unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg on the substrates of P2-P1. The isoelectric point is 5.2 by isoelectric focusing. N-Terminal sequencing gave a sequence of < EVPVEVVRXYVTSAPEK. The cysteine proteinase from Pacific cod very closely matches the previously reported miltpain from chum salmon.
Kawabata
C
C
Technical Research Center of T. Hasegawa Co., Ltd., Kawasaki, Japan. choko_kawabata@t-hasegawa.co.jp
Doi
Y
Y
Ichishima
E
E
D000595:Amino Acid Sequence; D000818:Animals; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005399:Fishes; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D003374:Coumarins; D004151:Dipeptides; D004791:Enzyme Inhibitors; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D003546:Cysteine Endopeptidases; C108050:miltpain