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

磷脂酶D（phospholipase D，PLD）对磷脂酰胆碱的水解是由多种外界信号引起的，目前认为PLD可产生多种脂质信使或介体，如甘油二酯，后者是蛋白激酶C（protein kinase C，PKC）激活所必需的。迄今为止，一些小分子量的G蛋白和蛋白磷酸化可能参与了PLD的激活，但酶的确切调控机制仍有待阐明。在本系列研究中，我们发现佛波酯或甘油二酯以ATP-Mg^<2+>依赖的方式增强了链溶素-O-透化的HL-60细胞中GTP γ S依赖的PLD活性。这种增强被完全抑制，不仅由PKC抑制剂，但酪氨酸激酶抑制剂表明蛋白激酶C和酪氨酸kinase.In无细胞系统的参与，GTP γ S是必不可少的PLD活性使用脾裂解物。我们还发现，一种新的蛋白质因子以外的G蛋白是必要的小分子量G蛋白（ADP核糖基化因子或Rho）的行动。这种新蛋白的纯化和表征正在研究中。

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.