Kasinathan C, Xu Z C, Kirchberger M A
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, NY 10029.
Lipids. 1989 Sep;24(9):818-23. doi: 10.1007/BF02544590.
Phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase activities in plasma membranes isolated from canine left ventricle were partially characterized, and their sensitivity to a number of intracellular variables was established. PtdIns and PtdIns4P kinase activities were estimated by the formation of [32P]PtdIns4P and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PtdIns(4,5)P2), respectively, when membranes were incubated with [gamma-32P]ATP and 0.1% Triton X-100. Unlike [32P]PtdIns4P formation [32P]PtdIns(4,5)P2 formation required exogenous (PtdIns4P) substrate. [32P]PtdIns4P and [32P]PtdIns(4,5)P2 formation were insensitive to Ca2+ at concentrations ranging from 0.1-30 microM. The hydrolysis of [32P]PtdIns4P was less than 15% under standard assay conditions for measuring its formation, and was unaffected by any of the variables tested. The apparent Km of the PtdIns kinase for ATP was 53 +/- 13 (S.E.M.) microM (N = 3). ADP inhibited [32P]PtdIns4P formation competitively with respect to ATP, the Ki being 0.4 mM. The data indicate that ADP is a poor competitive inhibitor of PtdIns kinase at the concentrations which are believed to be present intracellularly normally or which may be attained during mild hypoxia provided ATP levels are maintained in the millimolar range. Hence, any response of the myocardium to alpha-adrenergic hormones during mild hypoxia would be largely unimpaired by effects of Ca2+ on PtdIns and PtdIns(4,5)P2, or of ADP on PtdIns kinase activity.
对从犬左心室分离出的质膜中的磷脂酰肌醇（PtdIns）和磷脂酰肌醇4 - 磷酸（PtdIns4P）激酶活性进行了部分表征，并确定了它们对多种细胞内变量的敏感性。当膜与[γ - 32P]ATP和0.1% Triton X - 100一起孵育时，分别通过[32P]PtdIns4P和[32P]磷脂酰肌醇4,5 - 二磷酸（[32P]PtdIns(4,5)P2）的形成来估计PtdIns和PtdIns4P激酶活性。与[32P]PtdIns4P形成不同，[32P]PtdIns(4,5)P2形成需要外源性（PtdIns4P）底物。在0.1 - 30 microM的浓度范围内，[32P]PtdIns4P和[32P]PtdIns(4,5)P2形成对Ca2+不敏感。在测量[32P]PtdIns4P形成的标准测定条件下，[32P]PtdIns4P的水解小于15%，并且不受任何测试变量的影响。PtdIns激酶对ATP的表观Km为53±13（标准误） microM（N = 3）。ADP相对于ATP竞争性抑制[32P]PtdIns4P形成，Ki为0.4 mM。数据表明，在被认为正常存在于细胞内或在轻度缺氧期间可能达到的浓度下，只要ATP水平维持在毫摩尔范围内，ADP就是PtdIns激酶的一种弱竞争性抑制剂。因此，在轻度缺氧期间，心肌对α - 肾上腺素能激素的任何反应在很大程度上不会受到Ca2+对PtdIns和PtdIns(4,5)P2的影响，或ADP对PtdIns激酶活性的影响。
