Purification And Characterization Of Myofibril-bound Proteinases From The Muscle Of Basketshell Nassarius Siquinjorensis

Basketshell belongs to carnivorous species of the gastropod mollusk, which plays an important role in the benthic cenosis. Using the muscle of basketshell Nassarius siquinjorensis, the present paper studied the mechanism of its myofrbril autolysis. After a series of procedures, two myofibril-bound proteinases, a metalloproteinase and a serine proteinse, were isolated and characterized. In addition, endogenous serine proteinase inhibitors in basketshell muscle were preliminarily investigated as well.Significant autolysis of myobifrillar proteins of basketshell Nassarius siquinjorensis was observed during 55°C incubation as myosin heavy chain almost disappeared after 30 min, paramyosin significantly degraded into lower molecular mass polypeptides, and actin slightly degraded. Autolysis was partially inhibited by either 1, 10-phenanthroline or Benzamidine and almost completely by combination of EDTA and Benzamidine. It was proposed that the hydrolysis is quite possibly caused by combination of myofibril-bond metalloproteinase and serine proteinase.To purify the myofibril- bound metalloproteinase, heat extraction and successive chromatographies on DEAE-Sepharose, SP-Sepharose, and Phenyl- Sepharose were performed. At last, a metalloproteinase was isolated and named MBMP in brief. The molecular mass of the metalloproteinase was estimated to be 45 kDa with optimum pH and temperature of 8.0 and 60°C, respectively. The myofibrillar protein hydrolyzing activity of MBMP could be completely inhibited by EDTA,EGTA and 1, 10-phenanthroline while inhibitors of other proteinases showed no observable effect. Nevertheless, in order to purify the myofibril-bound seriene proteinase, heat extraction, ammonium sulfate fractionation and successive chromatographies on Sephacryl S-300, hydroxyapatite and SP-Sepharose were carried out. Finally, a serine proteinase was partially purified and thus named MBSP in brief. Under nonreducing condition the serine proteinase mainly migrated as a band of 21 kDa on SDS-PAGE stained with commassie blue R250. Its optimum pH and temperature were of pH8.0-9.0 and 55℃using mudsnail myofibrillar proteins as substrate, and of pH 9.0 and 40℃using Boc-Phe-Ser-Arg-MCA as flurescent substrate. In addition, endogenous serine proteinase inhibiting activity also presented in the heat extract which could depressed Boc-Phe-Ser-Arg-MCA hydrolyzing activities of either MBSPs from basketshell or crucian carp or porcine trypsin to some extent.