Studies on the coupling mechanism of phosphatidyl-choline turnover and protein kinase C activation.

磷脂酶D(PLD)对磷脂酰胆碱的水解是由多种外部信号引起的，目前认为它能产生多种脂质信使或介质，如对蛋白激酶C(PKC)激活至关重要的甘油二酯。到目前为止，一些小分子G蛋白和蛋白磷酸化可能参与了PLD的激活，尽管酶调节的确切机制仍未阐明。在本系列研究中，我们发现，在链溶素-O通透性的HL-60细胞中，磷脂或二酰甘油以依赖于ATP的方式增强了GTPRGammaS依赖的PLD活性。这种增强不仅被PKC抑制剂完全抑制，而且被酪氨酸激酶抑制剂完全抑制，表明参与了蛋白激酶C和酪氨酸激酶的参与。在一个无细胞的系统中，GTP-GammaS对于使用脾裂解物的PLD活性是必不可少的。我们还发现，小分子G蛋白(ADP核糖化因子或RHO)的作用需要除G蛋白之外的一种新的蛋白因子。这一新蛋白的纯化和性质正在研究中。

The hydrolysis of phosphatidylcholine by phospholipase D (PLD) is caused by a wide variety of external signngals, and is now thought to generate several lipid messengers or mediators such as diacylglycerol which is essential to protein kinase C (PKC) activation. To date, some of small molecular weight G-proteins and protein phosphorylation may be involved in the activation of PLD,although precise mechanism of the enzyme regulation remains to be elucidated. In the series of the present research, we have found that phorbolester or diacylglycerol enhanced GTPRgammaS-dependent PLD activity in an ATP-Mg^<2+> -dependent manner in streptolysin-O-permeabilized HL-60 cells. This enhancement was totally inhibited not only by PKC inhibitors but by tyrosine kinase inhibitors suggesting the involvement of protein kinase C and tyrosine kinase.In a cell-free system, GTPgammaS was essential to PLD activity using spleen lysates. We also found that a novel protein factor other than G-protein is necessary for the action of small molecular weight G-proteins (ADP ribosylation factor or Rho). Purification and characterization of this novel protein is under investigation.