Ando Y, Imamura S, Owada M K, Kakunaga T, Kannagi R
Department of Dermatology, Faculty of Medicine, Kyoto University, Japan.
Biochem Biophys Res Commun. 1989 Sep 15;163(2):944-51. doi: 10.1016/0006-291x(89)92313-9.
The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.
用钙离子载体A23187刺激人表皮样癌A431细胞，可导致高分子量脂皮质素I的形成，用特异性抗脂皮质素I抗体检测时，其表观分子量为75 kDa和160 kDa。这些免疫反应性蛋白被鉴定为脂皮质素I的共价交联多聚体，因为当纯化的脂皮质素I在体外与组织转谷氨酰胺酶（TGase）孵育时，观察到基本相同的交联多聚体。TGase的经典胺类底物，如丹磺酰尸胺和腐胺，也以化学计量方式掺入脂皮质素I中。脂皮质素II未观察到交联或胺掺入现象。缺失1 - 26位氨基酸的脂皮质素I不能作为TGase的底物，这表明脂皮质素I的N端区域在脂皮质素I多聚体的形成中起重要作用。TGase介导的脂皮质素I交联导致其对磷脂结合的钙敏感性显著增强；即，交联的脂皮质素I达到与磷脂酰丝氨酸囊泡最大结合量的50%所需的游离钙浓度低至3 microM，而完整的单体脂皮质素I则需要20 microM。
