title: Purification and characterization of gelatinase-like proteinases from the dark muscle of common carp (Cyprinus carpio).
abstract: Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Xiamen, China 361021.
authors.forename: Jiu-Lin
authors.initials: JL
authors.lastname: Wu
authors.forename: Bao-Ju
authors.initials: BJ
authors.lastname: Lu
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Du
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.forename: Ken-Ji
authors.initials: KJ
authors.lastname: Hara
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
mesh_terms: D000818:Animals; D002347:Carps; D002845:Chromatography; D003094:Collagen / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005590:Fractional Precipitation; D005780:Gelatin / Q000378:metabolism; D018093:Gelatinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D005780:Gelatin; D003094:Collagen; D018093:Gelatinases