草蝦血液中凝血系統及其酵素之研究

Abstract

自草蝦血球lysate，利用Fractogel TSK DEAE-650(M)及Sephacryl S-300管柱層析，分離Transglutaminase( TGase)，證實它是草蝦凝血所必需。此酵素在pH 7-9及25℃以下較穩定；其活性有賴Ca2+的存在，且受thiol reagents(例如PHMB, iodoacetamide), histamine及Zn2+之抑制，又可使primary amine (例如monodansylcadaverine)接到succinylaed casein上，這些性質皆和哺乳類的Factor XⅢa及liver TGase相似；但草蝦的TGase對Ca2+之需求非常嚴格(Sr2+，Ba2+，Mg2+皆無法取代)，且在螢光測定法不會有lag phase出現，又和plasma Factor XⅢa不同。草蝦血球的TGase之分子量在SDS-PAGE顯示91,500±3,000而gel filtration則顯示184,000，因此可能是以dimer存在，此分子量相似於文獻上所提lobster musule TGase (200,000)。Ca2+之存在使TGase活化也使之受thiol reagents inactivated的速度加快，推測可能是Ca2+使酵素conformation改變；另外，草蝦血球的TGase在各種情況下易失去活性，在有Ca2+的情況下尤然。 由草蝦plasma中，利用Sephacryl S-300及Fractogel TSK HW-65多次進行gel filtration後部分純化之 clottable protein(s)以SDS-PAGE分析有三個bands(分子量分別為207 K, 182K及164K)和文獻所言龍蝦之 fibrinogen monomer(分子量為190K)頗為相似。此部分純化的可凝蛋白加草蝦hemocyte lysate及Ca2＋，則有次序的逐漸凝集成巨大分子。而草蝦之凝血系統和人類、馬蹄蟹不同，可能不需要特別的蛋白?（如trypsin類）的參與。

Transglutaminase (TGase) was isolated from shrimp (Penaeus monodon) hemocyte lysate by Fractogel TSK DEAE-650(M) and Sephacryl S-300 chromatography. This enzyme was shown to be essential for the shrimp hemolymph clotting system. Its activity required Ca2+ and it was more stable at pH 7-9 and temperature below 25℃. Thiol reagents(for example, PHMB and iodoacetamide),histamine and Zn2+ inhibited the enzyme. The enzyme catalyzed the incorporation of primary amine such as monodansylcadaverine into succinylated casein and there was no initial lag phase in the fluorescence assay. The Ca2+ requirement for the shrimp hemocyte TGase was very stringent, Sr2+,Ba2+, Mg2+ can't substitute for Ca2+. The molecular weight of the shrimp hemocyte TGase was estimated to be 91,500 ±3,000 by SDS-PAGE, and 184,000 by gel filtration; thus the enzyme probably exists as dimers. Inactivation of the shrimp TGase by the thiol reagents was facilitated by Ca2+ and the activity of the enzyme was very unstable itself, especially in the presence of Ca2+. It was suggested that Ca2+ induced a conformational change. All these results suggest that the shrimp hemocyte TGase is similar to the mammalian platelet Factor XⅢa and liver TGase in many ways. Shrimp plasma clottable protein(s) was partially purified by Sephacryl S-300 and Fractogel TSK HW-65 gels. It gave three bands on SDS-PAGE corresponding to molecular weight 207,000, 182,000 and 164,000. The molecular weight is similar to that for the lobster fibrinogen subunit. After addition of the hemocyte lysate and Ca2+, the three bands disappeared on SDS-PAGE and could not enter the gel. However, the shrimp hemolymph clotting system appears not involve special proteinases of trypsin family.