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Four gelatinolytic proteinases (A1, 97 kDa; A2, 66 kDa; A3, 30 kDa; A4, 23 kDa) were detected from the red stingray wing muscle by gelatin zymography. Among these proteinases, major gelatinolytic proteinases (A1, A2) were more purified and examined their characters. The optimum pH and temperature for the gelatinolytic activity of A1 and A2 were around pH 7 and pH 8, and maximal activity of A1 is at 30-50C, that of A2 is at 30C. Also, they were demonstrated to hydrolyze the gelatin from red stingray; protein bands were almost completely degraded after 6 h incubation at 37C and pH 7.5. Both enzymes were strongly inhibited by Pefabloc SC, which suggests that they belong to gelatinolytic serine protease. PRACTICAL APPLICATIONS Gelatinolytic enzyme hydrolyzes denatured collagen such as gelatin. The enzymes from red stingray may be utilized to produce gelatin peptide, which has been widely used as additive to enhance the elasticity, consistency and stability of food products. In addition, it is meaningful in that this study will give information to understand the gelatinolytic proteinases in ray species and could lead to a comparative study of the biochemical properties of gelatinolytic proteinases in elasmobranch and many other species. [ABSTRACT FROM AUTHOR]

Abstract: Previously we have isolated a novel gelatinolytic serine proteinase, named G1, from the muscle and the plasma of red sea bream. In order to clarify the structure and function of G1, we cloned the full-length cDNA of G1 from the hepatopancreas of red sea bream. G1 cDNA encoded 633 amino acids with a secretory signal sequence at N-terminus, three epidermal growth factor-like domains, a kringle domain, and a trypsin-like serine protease domain. The active site residues of a serine proteinase were conserved in the serine protease domain of G1. The tissue distributions of the mRNA and gelatinolytic activity of G1 were investigated using RT-PCR and gelatin zymography, respectively. Its activity was detected in various tissues while the mRNA of it was strongly expressed in the hepatopancreas. These results suggest that G1 is synthesized in hepatopancreas and carried to the muscle, kidney, heart and ovary via the bloodstream in the red sea bream. The enzyme has a similar domain structure and tissue distribution to those of human hyaluronan binding protein 2 (HABP2) engaged in the extracellular matrix (ECM) turnover. Thus, it is suggested that G1 is identified as HABP2 and is possibly involved in ECM proteolysis of red sea bream. [Copyright &y& Elsevier]