Moritz A, De Graan P N, Gispen W H, Wirtz K W
Center for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
J Biol Chem. 1992 Apr 15;267(11):7207-10.
The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.
对从牛脑膜中纯化的磷脂酰肌醇-4-磷酸（PIP）激酶的脂质依赖性进行了研究。在所使用的测定方法中，将含有待测试脂质的PIP- Triton X-100胶束提供给该酶。在这些条件下，当达到PA与PIP的等摩尔比时，磷脂酸（PA）以浓度依赖性方式刺激酶活性，最高可达20倍。PA的刺激具有高度特异性；其他脂质，包括溶血磷脂酸和磷酸二鲸蜡酯，影响相对较小。PA的激活被磷脂酰肌醇4,5-二磷酸（PIP2）完全抑制。为了研究PA对天然膜中PIP激酶活性的影响，通过与磷脂酶D孵育在大鼠脑突触体细胞膜中产生内源性PA。随后用[γ-32P]ATP进行磷酸化，在这些膜中产生了PIP2而非PIP的增强标记。这些结果表明，PIP激酶活性可能受膜中PA水平的控制。这可能对激动剂诱导的磷酸肌醇周转调节细胞反应具有重要意义。
