PAF-STIMULATED PHOSPHOINOSITIDE TURNOVER IN PLATELETS

这位候选人对膜产生了永恒的兴趣， 磷酸肌醇（PPI）和磷脂酶。 近期目标是 为本申请中概述的研究项目奠定基础 从长远来看，这将使PPI的重要性得以揭示， 和磷脂酶在细胞功能中的作用 项目：磷脂酶C（PLC）在各种细胞中分解PPI， 包括血小板参与信号转导。 的假设 （a）血小板活化因子（PAF）受体通过 G蛋白对血小板PLC的激活和调节， （B）外源性PLC或内源性PLC的激活引起PI-1的释放， 血小板膜表面的锚定蛋白。 有四 实验计划 [I]以确定PAF受体偶联激活的机制， PLC的脱敏：（A）将[3 H]PAF结合与PLC相关联 与对照组相比，脱敏血小板的活化;（B）与对照组相比，脱敏血小板的活化 通过使用NaF、GTP γ和百日咳毒素操纵G蛋白， 关联它们对PLC活化的影响;（C）关联蛋白质 激酶C介导的磷酸化激活和脱敏 的PLC。 [II]确定PLC、G蛋白和PAF之间的分子相互作用 受体：血小板膜将被溶解和分离， 柱层析 [3 H]PAF结合、GTP酶、32 P-GTP结合和PLC 将对活动进行分段监测，以确定其功能 协会. [III]PAF受体偶联激活PLC的重要性 糖尿病患者血小板的超敏反应。 [IV]测定PI锚定膜的释放 外源性和内源性PLC的蛋白质/糖蛋白： 蛋白质（B.苏云金杆菌（thuringiensis）; 通过SDS-PAGE/荧光照相术和抗体鉴定;使用 微分相分离以监测PI锚定蛋白， PAF刺激。 该项目将提供PAF刺激PPI周转的新见解 以及血小板中PI锚定蛋白的重要性。

The candidate has developed an everlasting interest in membranes, phosphoinositides (PPI) and phospholipases. The immediate goal is to build a foundation for the research project outlined in this application which will enable, in the long term, to unravel the importance of PPI and phospholipases in cellular functions. Project: Breakdown of PPI by phospholipase C (PLC) in various cells, including platelets, is involved in signal transduction. The HYPOTHESIS is that (a) platelet activating factor (PAF) receptor is coupled via a G-protein to the activation and regulation of the PLC in platelets and (b)exogenous PLC or activation of endogenous PLC cause release of PI- anchored proteins from platelet membrane surfaces. There are four experimental plans. [I] TO DETERMINE MECHANISM(S) OF PAF RECEPTOR-COUPLED ACTIVATION AND DESENSITIZATION OF PLC: (A) to correlate [3H]PAF binding to PLC activation in desensitized platelets as compared to control; (B) to manipulate G-proteins by using NaF, GTPgammas and pertussis toxin and to correlate their influence on PLC activation; (C) to correlate protein kinase C-mediated phosphorylation to the activation and desensitization of PLC. [II] TO DETERMINE MOLECULAR INTERACTIONS AMONG PLC, G-PROTEINS AND PAF RECEPTOR: Platelet membranes will be solubilized and fractionated by column chromatography. [3H]PAF binding, GTPase, 32P-GTP binding and PLC activities will be monitored in fractions to establish their functional associations. [III] TO DETERMINE IMPORTANCE OF PAF RECEPTOR COUPLED ACTIVATION OF PLC IN THE HYPERSENSITIVITY OF DIABETIC HUMAN PLATELETS. [IV] TO DETERMINE THE RELEASE OF PI-ANCHORED MEMBRANE PROTEIN/GLYCOPROTEIN BY EXOGENOUS AND ENDOGENOUS PLC: Release of proteins by PLC (B. thuringiensis) from labelled platelets; identification by SDS-PAGE/fluirography and antibodies; use of differential phase partitioning to monitor PI-anchored proteins after PAF stimulation. The project will provide novel insight(s) in PAF-stimulated PPI turnover and on the importance of PI-anchored proteins in platelets.