PAF-STIMULATED PHOSPHOINOSITIDE TURNOVER IN PLATELETS

这位候选人对膜产生了持久的兴趣， 磷脂酶(PPI)和磷脂酶。眼下的目标是 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的重要性得到揭示 以及细胞功能中的磷脂酶。 项目：磷脂酶C(PLC)在不同细胞中分解PPI， 包括血小板在内，都参与信号转导。假说 是(A)血小板活化因子(PAF)受体通过 G蛋白对血小板内PLC的激活和调节 (B)外源性PLC或内源性PLC的激活导致PI的释放- 从血小板膜表面锚定的蛋白质。一共有四个 实验计划。 [I]确定PAF受体偶联激活的机制(S) PLC的脱敏作用：(A)[~3H]PAF与PLC结合的相关性 与对照组相比，脱敏血小板的激活； 利用NaF、GTP和百日咳毒素操纵G蛋白，并 关联它们对PLC激活的影响；(C)关联蛋白质 激酶C介导的磷酸化对激活和脱敏的影响 可编程逻辑控制器。 [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级 柱层析法。PAF结合、GTP酶、32P-GTP结合和PLC 将对部分活动进行监控，以确定其功能 联想。 [III]确定PAF受体偶联激活PLC的重要性 糖尿病人血小板的超敏反应。 [IV]测定PI锚定膜的释放度 外源性和内源性PLC的蛋白质/糖蛋白：释放 PLC(苏云金芽孢杆菌)从标记的血小板中提取蛋白质； 用十二烷基硫酸钠-PAGE/荧光照相法和抗体鉴定 差示相分割技术监测PI锚定蛋白的研究 PAF刺激。 该项目将为PAF刺激的PPI成交额提供新的见解(S) 以及PI锚定蛋白在血小板中的重要性。

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.