Regulation of phosphatidylinositol kinase activity

磷脂酰肌醇(PI)的转化被认为是受体介导的信号转导通路的重要组成部分，以响应各种激素和生长因子。在这种情况下，两个假定的第二信使，二酰甘油(DG)和肌醇1，4，5-三磷酸，是通过磷脂酰肌醇4，5-二磷酸的水解而产生的。DG激活蛋白激酶C，而1，4，5-三磷酸肌醇引起细胞内钙离子的释放。随着钙离子的释放，磷脂酰肌醇4，5-二磷酸被磷脂酰肌醇和磷脂酰肌醇4-磷酸(PIP)激酶逐步磷酸化，从而快速地重新合成磷脂酰肌醇4，5-二磷酸，从而提供产生第二信使的底物。为了阐明PI激酶在PI周转调节中的可能重要性，还需要对该酶进行纯化和性质鉴定。在本研究中，一种膜结合型pI KIN…的纯化和鉴定从大鼠脑中提取了更多的酶。从大鼠脑中纯化了一种膜结合的磷脂酰肌醇(PI)激酶。用盐洗膜中的Triton X-100溶解该酶，纯化倍数为11,183倍，最终比活力为150nmol/min/mg蛋白质。纯化步骤包括使用Q-SepharoseFast Flow、磷酸纤维素、Toyota opearl HW 55和Affi-Gel Blue进行几次层析。经凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得其相对分子质量分别为80,000和76,000。纯化后的蛋白只磷酸化PI，不磷酸化磷脂酰肌醇4-磷酸或甘油二酯。测得PI和ATP的K_m分别为115和150微米。该酶需要5-20 mM的镁离子或1-2 mM的锰离子，被0.1-1.0%(w/v)的Triton X-100刺激，而被0.05%的十二烷基硫酸钠完全抑制。该酶的最适PH值在7.4左右。该酶利用ATP而不是GTP作为磷酸盐供体。三磷酸腺苷和二磷酸以外的核苷三磷酸对该酶活性有明显的抑制作用。然而，腺苷、cAMP和槲皮素的抑制作用较弱。较少

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