Tokunaga F, Yamada M, Miyata T, Ding Y L, Hiranaga-Kawabata M, Muta T, Iwanaga S, Ichinose A, Davie E W
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1993 Jan 5;268(1):252-61.
To investigate further the molecular events of the intracellular coagulation cascade in limulus hemocytes, a transglutaminase (TGase), which may be involved in the formation of a stabilized gel, was purified and characterized. Through the purification procedures consisting of six steps, 1.6 mg of TGase with a specific activity of 940 amine incorporation unit/mg was obtained from 32.4 g of Tachypleus tridentatus hemocytes. The purified TGase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular mass of 86 kDa, and demonstrated mammalian-type II TGase-like enzymatic properties. The TGase activity was Ca(2+)-dependent and was inhibited by primary amines, EDTA, and SH-reagents. Moreover, two major potential substrates for TGase were identified in the hemocyte lysate by using dansylcadaverine (DCA) incorporation in the presence of 10 mM CaCl2 and 10 mM dithiothreitol. Of these protein substrates, an 80-kDa protein contained a large number of proline residues, amounting to about 22% of the total amino acids. On the other hand, an 8.6-kDa protein abundantly present in the hemocytes was characterized as a Cys-rich protein consisting of 81 amino acid residues and a calculated molecular mass of 8,671. The entire amino acid sequence of this protein was established. Also, the 8.6-kDa protein was readily cross-linked intermolecularly by TGase, forming multimers as large as pentamers. We speculate that like plasma factor XIIIa, limulus TGase and its two protein substrates in the hemocytes may play an important role in the defense of this animal against invading microorganisms.
为了进一步研究鲎血细胞内凝血级联反应的分子事件，一种可能参与稳定凝胶形成的转谷氨酰胺酶（TGase）被纯化并进行了特性鉴定。通过包含六个步骤的纯化程序，从32.4克三刺鲎血细胞中获得了1.6毫克比活性为940胺掺入单位/毫克的TGase。纯化后的TGase在SDS-聚丙烯酰胺凝胶电泳上呈现单一条带，分子量为86 kDa，并表现出哺乳动物II型TGase样的酶学特性。TGase活性依赖于Ca(2+)，并受到伯胺、EDTA和巯基试剂的抑制。此外，在10 mM CaCl2和10 mM二硫苏糖醇存在的情况下，通过丹磺酰尸胺（DCA）掺入法在血细胞裂解物中鉴定出了TGase的两种主要潜在底物。在这些蛋白质底物中，一种80 kDa的蛋白质含有大量脯氨酸残基，约占总氨基酸的22%。另一方面，血细胞中大量存在的一种8.6 kDa蛋白质被鉴定为富含半胱氨酸的蛋白质，由81个氨基酸残基组成，计算分子量为8671。确定了该蛋白质的完整氨基酸序列。此外，8.6 kDa蛋白质很容易被TGase进行分子间交联，形成高达五聚体的多聚体。我们推测，与血浆因子XIIIa一样，鲎TGase及其在血细胞中的两种蛋白质底物可能在这种动物抵御入侵微生物的防御中发挥重要作用。
