| Research Abstract |
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
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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
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