Research of the physiological role of marine mollusks cross -linking enzymes and structures of its substrates.

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Marine mollusk contains a transglutaminase (Tgase) which is activated by a contact with sea water or body fluid, suggesting it might be acted to stop bleeding by cross linking of proteins. To clarify the physiological role of Tease, identification of its intracellular substrates and common structural properties were investigatedThe main component of the intracellular substrate of scallop hemocyte, 45 kDa protein, was revealed to be Actin by amino-terminal sequences of BrCN-cleaved 15k and 12k fragments, YVAIQVLSL and GQKDSYVGDE, respectively.When carp myosin was incubated with Tgase and monodansylcadaverine MO, which is a fluorescent substrate of the enzyme, MDC was specifically incorporated in to S2 region of myosin heavy chain. B PLC analysis of the MDC-incorporated fragments derived from BrCN-cleavage and proteinase digestion showed that Gln(520) was the first cross-linking site in the myosin heavy chain. The Gln(520) was also identified to be the primary mss-linking site of Alaska pollack myosin. To compare the reactivity of scallop myosin heavy chain to those of fish ones, the complete amino acid sequence of scallop myosin heavy chain was determined by cDNA sequence analysis for the first time. The cross linking sites of scallop myosin were located in several Gln residues in the rod region containing Gln(520), suggesting the difference in the reactivity between substrate proteins.The intracellular substrates for squid gill Tgases were insoluble 190 kDa and 90 kDa proteins for Japanese common squid, and 190 kDa component for spear squid, respectively. Although the amino-terminal sequences of two BrCN-cleaved fragments of 190 kDa protein were determined to be AIDVE and ANTDEAL, respectively, no significant homology to the known proteins was detected. As the 200 kDa protein was also supposed to be a primary intracellular substrate for scallop hemocyte Tgase, these proteins were suggested to be common intracellular substrates for marine molluskan Tgases.