Purification And Characterization Of A Serine Proteinase Inhibitor From Skeletal Muscle Of Silver Carp (Hypophthalmichthys Molitrix) And Its Effect On The Proliferation Of Tumor Cells

Proteases control the hydrolysis of the protein, and protease activities are controlled by several means, including protease inhibitors. Most protease inhibitors can control the biological process by affecting the protease activity reversibly, so they had a wide application. The source of silver carp (Hypophthalmichthys molitrix) is rich in China, so it is necessary to utilize them. Surimi production is the best-known example of food processing with the aid of protease inhibitors. Substance which contains protease inhibitors can prevent the degradation of fish muscle. Some limitations of these inhibitor products are the off flavor and off color imparted to the final product. These are reasons why alternative inhibitor sources are currently studied. It is the first time to purify a serine proteinase inhibitor from the silver carp. The character, the sequence, the effect on the autolysis of myofibrillar proteins, inhibition on the tumor cell proliferation and cell circle of the inhibitor were studied.The serine proteinase inhibitor was purified to homogeneity from the skeletal muscle of silver carp skeletal muscle in this dissertation. Purification was carried out by chromatographies on DEAE-Sephacel, SP-Sepharose, Blue-Sepharose 6 Fast Flow, and Con A-Sepharose column. The specific activity of the inhibitor increased by 266 fold and the yield was about 6%. The purified inhibitor was a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE. Gelatin reverse zymography showed it inhibited trypsin activity. The thermal stability of the inhibitor considerably decreased at temperatures >60℃, and it only left 30% of its initial inhibitory activity when treatment above 70℃.The activity of the inhibitor increased in presence of K⁺, but Mg²⁺, Na⁺ and Ca²⁺ had no effect. The inhibitor showed a wide range of pH stability (from 3 to 11). It revealed high inhibition specificity toward serine proteinases (bovine trypsin, bovine chymotrypsin, and porcine trypsin). It did not exert any inhibitory activity toward cystein proteases (papain) and aspartyl proteinases (bovine pepsin). The inhibitor worked on the trypsin in a dose dependent manner. Inhibitor-trypsin complex was formed and a high molecular weight protein band could be revealed in the SDS-PAGE. The peptide profile was determined by peptide mass figerprint using trypsin digested. It showed no homology with known serine proteinase inhibitor. The N-terminus amino acid sequence was found by the Edman degradation sequencing method ARYEDHDCDG, and also showed no homology with known serine proteinase inhibitor.Myofibrillar proteins of silver carp remarkably degraded at 55℃. The inhibitor, PMSF, E-64 and EDTA inhibited the hydrolysis of the myofibrillar protein, especially the degradation of myosin heavy chain (MHC). Proteases or one kind of myofibril-bound serine proteinase was deduced to work on the hydrolysis of the myofibrillar protein. The hydrolysis of the myofibrillar protein by the extesic trypsin could be inhibited by addtion of the silver carp inhibitor completely.The antiproliferation of tumor cells by treatment of the purified silver carp inhibitor was determined with MTT on Sp20 cells, Jarkat cells and MG63 cells. Results showed the inhibition on the proliferation for the three cell lines in dose-dependent manner. Concentration of 0.1 g/L of silver carp inhibitor hadeffect on the Jarkat cells. The inhibitor arrested the cell cycle at G₀/G₁ phase of Jarkat cells afeter the silver carp inhibitor treatment for 8h. No apoptosis of Jarkat cells was observed after treatment for 24h.