Abstract
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 phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (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.
Highlights
Followingthe breakdown, phosphatidylinositol 4,5-bisphosphate is rapidly resynthesized by stepwise phosphorylation of PI by PI kinase and phosphatidylinositol 4-phosphate (PIP) kinase, and the substrate for the generation of second messengers is continuously supplied
-00..22\ pp tions from the Q-Sepharose Fast Flow column were mixed with 100 ml of buffer C (20 mM Hepes/NaOH, 1mM EDTA, 1mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and10% glycerol) and applied to a cellulose phosphate column (2.5 X 30 cm) preequilibrated with buffer C
The membrane-bound PI kinase was mostly extracted by Triton X-100 treatment from the salt-washed membranes, and the yield of this step was usually more than loo%, and, the yield of the Q-Sepharose step sometimes exceeded 100%.These resultssuggestthat some kind of intrinsic inhibitor may be removedat these steps.the yield was calculated andpresented on the basis that the total activity of the Tritonextract was 100%
Summary
The abbreviations used are: PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; DG, diacylglycerol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonicacidDTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; protein kinase C, Ca"/phospholipid-dependent enzyme. Protein kinase C activation by tetradecanoylphorbol acetate or oleoylacetylglycerol enhances PIP synthesis from PI [10, 11].a close relationship between activation of PI kinase and enhanced PI turnover has been demonstrated in fibroblasts under growth factor stimulation [12, 13], or viral transformation [14,15,16,17] These changes of PI kinase activity are considered to be caused through protein phosphorylation reactions, but the precise mechanism is not clear. The resultant lower phase was removed into a vial and dried, and theradioactivity was measured by liquid scintillation counting
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