Regulation of phospholipase D from human hepatocarcinoma cell line by purine nucleotides and protein kinase A

The regulation of phosphatidylcholine-specific phospholipase D by purine nu cleotides and protein kinase A were studied in vitro using an enzyme prepar ation partially purified from the membranous fraction of 7721 hepatocarcino ma cells. It was found that the enzyme activity was elevated by low concent rations of some purine nucleotides, but the activating effects were decreas ed when the concentrations of the nucleotides were higher. The optimal conc entrations of GTP, GTP gamma[S] , GDP and ATP for maximal activation were 0 .1mM, 5 mu M,1 mM and 1 mM respectively. The activation caused by 1mM ADP w as lower. The enzyme was not activated by 1mM AMP, but significant activati on was observed by the addition of 1mM cAMP. The latter was mediated by pro tein kinase A, as a specific inhibitor of protein kinase A ablished the act ivation. There were synergic effects between ATP and GTP, ATP and PIP2, but not between ATP and GTP gamma[S] , or PIP2 and GTP gamma[S]. The activatin g effects of GTP and ATP were abolished by neomycin, a PIP2 scavenger. Thes e results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP2 is required for the activation induced by GTP. Pro tein kinase A may be another protein kinase in addition to protein kinase C and protein tyrosine kinase which regulate the activity of phospholipase D , when the intracellular concentration of cAMP is increased.