Study Of An Endogenous Inhibitor To Myofibril-bound Serine Proteinase From Crucian Carp (Carassius Auratus)

The modori phenomenon (thermal gel degradation of fish jelly products) during the surimi production is an important factor for the quality of surimi. Compared with marine fish, fresh water fish surimi gel was prone to degradation more easily. The evidence of the degradation of myofibrillar protein by myofibril-bound serine proteinase (MBSP) was the major reason for modori phenomenon. Therefore, the search for a natural inhibitor to MBSP become another direction in the studies of surimi processing. Recently, we have identified that a specific endogenous inhibitor toward MBSP in the skeletal muscle of marine fish white croaker (Argyrosomus argentatus) had a high degree of homology with porcine glucose-6-phosphate isomerase (GPI). However, the MBSPI on fresh fish has not been reported, in the present, we described a detailed study on the MBSPI from the skeletal muscle of crucian carp with an attempt to clarify the inhibition mechanism between GPI and MBSP.MBSP was purified from the skeletal muscle of crucian carp by buffer extraction, acid precipitation, Q-Sepharose ion exchange chromatography, and Benzamidine Sepharose affinity chromatography with a yield of 2.4 % and purification of folds of 600. Substrate specificity analysis showed that MBSP can specifically degraded trypsin type substrates, and the inhibitor susceptilibility analysis revealed that MBSP was inhibited effectively by serine proteinase inhibitor, the cysteine proteinase inhibitor E-64 had some degree inhibition on MBSP, indicated MBSP is a trypsin like serine proteinase.Glucose-6-phosphate isomerase was purified to homogeneity from the skeletal muscle of crucian carp by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose and Superdex 200 with a yield of 8.0 % and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration using Superdex 200 column, while on SDS-PAGE, two subunits (55 kDa and 65 kDa) were identified, suggesting it is quite possibly a heterodimer. The GPI also digested by trypsin and analyzed by LC-MS/MS, the result showed that it had a 91.7 % and 97.5 % homology with zebra fish gpia and gpib, respectively. Interestingly, GPI revealed specific inhibitory activity toward myofibril-bound serine proteinase (MBSP) from crucian carp while no inhibitory activity was identified toward other serine proteinases such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP and the Ki was 0.075μmol/L. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo. The possible application of GPI as an inhibitor to MBSP in surimi preparation was also discussed.