title: Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).
abstract: A gelatinolytic matrix metalloproteinase (gMMP) from grass carp skeletal muscle was purified by 30-70% ammonium sulphate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, and affinity on gelatin-sepharose. The molecular weight of the proteinase as estimated by SDS-PAGE was 70 kDa under non-reducing conditions. The enzyme revealed high activity from 30 to 50 °C, and the gelatin hydrolysing activity was investigated at a slightly alkaline pH range using gelatin as substrate. Metalloproteinase inhibitor EDTA completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was essential for the gelatinolytic activity. Further, peptide mass fingerprinting obtained four fragments with 45 amino acid residues, which were highly identical to MMP-2 from fish species. The gMMP could effectively hydrolyse type I collagen even at 4 °C, suggesting its involvement in the texture softening of fish muscle during the post-mortem stage.
authors.affiliation: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002, China. Electronic address: wujiul590@126.com.
authors.forename: Jiu-Lin
authors.initials: JL
authors.lastname: Wu
authors.forename: Shang-Ying
authors.initials: SY
authors.lastname: Ge
authors.forename: Zhi-Xing
authors.initials: ZX
authors.lastname: Cai
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Ying-Xiang
authors.initials: YX
authors.lastname: Liu
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Wang
authors.forename: Qi-Qing
authors.initials: QQ
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry; D002347:Carps; D002850:Chromatography, Gel; D024042:Collagen Type I / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020782:Matrix Metalloproteinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D024042:Collagen Type I; D010455:Peptides; D011480:Protease Inhibitors; D005780:Gelatin; D004492:Edetic Acid; D020782:Matrix Metalloproteinases; D002118:Calcium
keywords: Gelatin zymography; Grass carp; Matrix metalloproteinase; Post-mortem tenderization; Purification