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Miltpain, a cysteine proteinase, from milt of Pacific cod (Gadus macrocephalus): purification and characterization
C Kawabata1, Y Doi, E Ichishima
1Technical Research Center of T. Hasegawa Co., Ltd., Kawasaki, Japan. choko_kawabata@t-hasegawa.co.jp
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 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.
Insights
A novel cysteine proteinase, similar to chum salmon miltpain, was identified in Pacific cod milt. This enzyme preferentially hydrolyzes basic proteins and exhibits unique substrate specificity.
Area of Science:
- Biochemistry
- Enzymology
- Marine Biology
Background:
- Miltpain, a cysteine proteinase, was previously identified in chum salmon milt.
- Cysteine proteinases play crucial roles in various biological processes.
Purpose of the Study:
- To isolate and characterize a similar cysteine proteinase from Pacific cod milt.
- To compare the properties of the cod enzyme with known miltpains.
Main Methods:
- Enzyme isolation and purification using gel filtration and SDS/PAGE.
- Enzymatic activity assays with Z-Arg-Arg-MCA substrate.
- Inhibition studies with various protease inhibitors.
- Isoelectric focusing and N-terminal sequencing.
Main Results:
- A cysteine proteinase was purified 6900-fold from Pacific cod milt.
- The enzyme has an estimated mass of 63-72 kDa, optimal pH of 6.0, and specific kinetic parameters (Km, kcat).
- The proteinase hydrolyzes basic proteins and shows specificity for paired basic residues, closely matching chum salmon miltpain.
Conclusions:
- Pacific cod milt contains a cysteine proteinase homologous to chum salmon miltpain.
- The characterized enzyme possesses distinct biochemical and substrate specificity properties.