Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization |
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Authors: | Kawabata C Doi Y Ichishima E |
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Institution: | a Technical Research Center of T. Hasegawa Co., Ltd., Kariyado, 335 Nakahara-ku, Kawasaki 211-0022, Japan;b Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan |
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Abstract: | 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 μM 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. |
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Keywords: | Cathepsin Cod Cysteine protease Gadus macrocephalus Miltpain Noncompetitive inhibition Pacific cod o-Phenanthroline |
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