Identification Of A Metalloproteinase From Blue Scad(decapterus Maruadsi) Muscle And Its Effect On Collagen

Metalloproteinases are enzymes that their active sites are composed of metal ions and animo acid residues. Matrix metalloproteinases(MMPs) are metal ion-dependent proteinases that are largely responsible for degrading ECM proteins. MMPs have been proposed to participate in the post-mortem degradation of fish muscle. However, there have seldom reports about MMPs from saltwater fish muscle on protein level. In the present study, using blue scad(Decapterus maruadsi) as material, we tried to study enzymes that can degradate collagen, and then further to discuss the effect of matrix metalloproteinases on collagen.By gelatin zymography, a metalloproteinase from the skeletal muscle of blue scad which may responsible for the degradation of collagen was identified. The proteinase was further purified to high purity by ammonium sulfate fractionation and chromatographies on DEAESephacel, Phenyl-Sepharose and Gelatin-Sepharose. The molecular mass of the metalloproteinase were 57 k Da and 55 k Da as estimated by SDS-PAGE. It revealed high activity at a slightly alkaline p H range and the optimum temperature was 40℃. Metalloproteinases inhibitors such as EDTA, EGTA and 1,10-phenanthroline suppressed the activity specifically. Metal ions such as Ca2+, Ba2+and Zn2+ activated the enzyme obviously, especially Ca2+.The collagen was extracted by pepsin digestion and fractionated by salt precipitations into type I collagen and type V collagen. For type V collagen, Peptide mass fingerprinting revealed that the protein was identical to 130 amino acid residues with 100% identical to collagen alpha-2(V) chain precursor(gi:224809395) from Danio rerio, strongly suggesting the purified protein is type V collagen. Purified type V collagen was used as antigen to injected subcutaneously into the rabbit, which successfully produce polyclonal antibody. The specificity of anti-collagen(V) polyclonal antibody is superior which confirmed by western blotting. The denaturation temperature of type V collagen(30.5℃) was significantly higher than type I collagen(28.5℃) assessed by circular dichroism(CD). Type I and V collagens were prepared from common carp muscle as substrates. The metalloproteinase hydrolyzed type I and V collagen effectively at 37 ℃and even 4℃, strongly suggesting its involvement in the proteolytic breakdown of collagens in fish muscle during postmortem tenderization. Furthermore, the metalloproteinase effectively hydrolyze native collagen.This manuscript constructed a series of process to purify the metalloproteinase. The impact of temperature, p H, proteinase inhibitors and metal ions on the activities of enzymes was also discussed. we tried to study the effect of matrix metalloproteinases on collagen. This study is helpful for understanding the biochemical role in fish muscle and during the post-mortem tenderization of fish muscle.