Hanigan H, Goldsmith L A
Biochim Biophys Acta. 1978 Feb 10;522(2):589-601. doi: 10.1016/0005-2744(78)90090-6.
Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.
表皮转谷氨酰胺酶在体内的潜在底物已在人类角质层和新生大鼠表皮中分离出来，并进行了部分特性鉴定。[14C]腐胺和丹磺酰尸胺在体外被掺入表皮蛋白中。在大鼠和人类匀浆中，有两种高分子量蛋白掺入了这些标记物。其中一种蛋白太大，无法进入4%十二烷基硫酸钠-聚丙烯酰胺间隔胶；另一种则出现在间隔胶与10%十二烷基硫酸钠-聚丙烯酰胺电泳胶的界面处。这些蛋白存在于缓冲液提取物、十二烷基硫酸钠-二硫苏糖醇提取物和氢氧化钠提取物中。在上述提取后剩余的不溶性沉淀中的蛋白也掺入了标记物。腐胺和丹磺酰尸胺的掺入是时间依赖性的，并受到表皮转谷氨酰胺酶已知抑制剂的抑制。这两种高分子量蛋白具有相似的氨基酸组成，其特点是甘氨酸、谷氨酸、丝氨酸和天冬氨酸含量高。氨基酸组成与α-角蛋白相似，但不完全相同。在转谷氨酰胺酶存在下孵育的表皮匀浆显示蛋白逐渐不溶。这种交联被腐胺抑制。[14C]甘氨酸、[14C]组氨酸和[4C]脯氨酸在新生大鼠体内被掺入表皮蛋白中。在转谷氨酰胺酶存在下体外孵育时，甘氨酸标记的蛋白变得越来越不溶。转谷氨酰胺酶的体外孵育对组氨酸和脯氨酸标记的蛋白没有影响。
