Regulation of phosphatidylinositol kinase activity

磷脂酰肌醇（PI）的转换被认为是响应各种激素和生长因子的受体介导的信号转导途径的重要组成部分。在这种情况下，两个假定的第二信使，二酰基甘油（DG）和肌醇1,4,5-三磷酸，是通过磷脂酰肌醇4,5-二磷酸水解产生的。DG激活蛋白激酶C，而肌醇1,4,5-三磷酸引起细胞内钙储存中Ca^<2+>的释放。分解后，磷脂酰肌醇4,5-二磷酸通过PI激酶和磷脂酰肌醇4-磷酸（PIP）激酶的逐步磷酸化迅速重新合成，从而持续提供第二信使生成的底物。为了阐明PI激酶在PI转换调控中的可能重要性，还需要对该酶进行纯化和表征。本研究从大鼠脑中分离纯化了一种膜结合形式的PI kin…More酶。从大鼠脑中纯化了一种膜结合的磷脂酰肌醇激酶。用triton X-100从盐洗膜中溶解该酶，得到11,183倍纯化，最终比活性为150 nmol/min/mg蛋白质。纯化步骤包括使用Q-Sepharose Fast Flow、磷酸纤维素、Toyopearl HW 55和Affi-Gel Blue进行层层析。纯化后的PI激酶经凝胶过滤估计分子量为80,000，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计分子量为76,000。纯化后的激酶仅磷酸化PI，而不磷酸化磷酸磷脂酰肌醇4-磷酸或二酰基甘油。PI和ATP的K_m值分别为115和150 muM。该酶需要Mg^<2+>(5-20 mM)或Mn^<2+>(1-2 mM)才能产生活性，0.1-1.0% (w/v) Triton X-100刺激该酶，0.05%十二烷基硫酸钠完全抑制该酶。酶活性显示，PH值在7.4左右时最适。该酶利用ATP而不是GTP作为磷酸供体。除ATP和二磷酸外，三磷酸核苷显著抑制激酶活性。而腺苷、cAMP和槲皮素的抑制作用较弱。少

Phosphatidylinositol (PI) turnover has been thought to be an essential part of receptor-mediated signal transduction pathways in response to various hormones and growth factors. In this case, two putative second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate, are generated through the hydrolysis of phosphatidylinositol 4,5-bisphosphate. DG activates protein kinase C, whereas inositol 1,4,5-trisphosphate causes the release of Ca^<2+> from an intracellular calcium store.Following the breakdown, phosphatidylinositol 4,5-bisphosphate is rapidly resynthesized by stepwise phosphorylation of PI by PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase, and thus the substrate for the generation of second messengers is continuously supplied.To clarify the possible importance of PI kinase in the regulation of PI turnover, purification and characterization of the enzyme is also required. In this study, the purification and characterization of a membrane-bound form of PI kin … More ase from rat brain was carried out.A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylated phosphatidylinositol 4-phosphate or diacylglycerol. K_m values for PI and ATP were found to be 115 and 150 muM, respectively. The enzyme required Mg^<2+>(5-20 mM) or Mn^<2+>(1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad PH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak. Less