Tyrosyl and phosphatidylinositol kinases of human erythrocyte membranes.
Tyrosyl and phosphatidylinositol kinases of human erythrocyte membranes.
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人红细胞膜的酪氨酰和磷脂酰肌醇激酶。
DOI：
10.1002/jcb.240330402
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发表时间：
1987
影响因子：
4
通讯作者：
Macara,IG
中科院分区：
文献类型：
--
作者：
Vossler,MR;Coco,A;Strausser,BT;Zaricznyj,C;Macara,IG
人红细胞的酪氨酰激酶和磷脂酰肌醇 (PI) 激酶活性已部分纯化和表征。尽管PI激酶需要去污剂来溶解，但红细胞的主要酪氨酰激酶可以通过高盐提取。然而，膜上仍然存在非常小的残留活性，该活性被 PI 激酶溶解，并通过硫酸铵沉淀和二乙氨基乙基 (DEAE) 离子交换步骤梯度洗脱进行共纯化。然而，我们发现这两种活性在 ATP 和 Mg2+ 的表观 Kms 方面存在差异；它们表现出不同的温度失活半衰期，在 Mn+ 和 Ca2+ 存在下具有不同的相对活性，并且可以通过使用线性 NaCl 梯度从 DEAE-Trisacryl 离子交换柱洗脱来分离。膜相关酪氨酰激酶的动力学参数与盐提取酶的动力学参数不同。通过向完整膜添加磷酸酪氨酰磷酸酶抑制剂钒酸盐进行酪氨酰激酶 p68v-rosor 预处理，PI 激酶不会被激活，并且不会被酪氨酰激酶底物聚 (Glu4,Tyr) 竞争性抑制。我们得出的结论是，人红细胞磷脂酰肌醇和酪氨酰激酶具有不同且可分离的活性，并且人红细胞中至少存在两种可分离的酪氨酰激酶。
The tyrosyl kinase and phosphatidylinositol (PI) kinase activities of human red cells have been partially purified and characterized. Although the PI kinase required detergent for solubilization, the major tyrosyl kinase of the red cell could be extracted by high salt. A very small residual activity remained associated with the membranes, however, that was solubilized with the PI kinase and copurified through an ammonium sulfate precipitation and diethylaminoethyl (DEAE) ion‐exchange step gradient elution. However, the two activities were found to differ with respect to their apparent Kms for ATP and Mg2+; they showed different half‐lives for temperature inactivation, possessed different relative activities in the presence of Mn+and Ca2+, and were separable by elution from a DEAE‐Trisacryl ion exchange column using a linear NaCl gradient. The kinetic parameters of the membrane‐associated tyrosyl kinase differed from those of the salt‐extracted enzyme. PI kinase was not activated by pretreatment with the tyrosyl kinase p68v‐rosor by addition of the phosphotyrosyl phosphatase inhibitor, vanadate, to intact membranes, and was not competitively inhibited by the tyrosyl kinase substrate poly(Glu4,Tyr). We conclude that the human red cell phosphatidylinositol and tyrosyl kinases are distinct and separable activities, and that at least two separable tyrosyl kinases are present in human erythrocytes.