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 nucleotides and protein kinase A were studied in vitro using an enzyme preparation partially purified from the membranous fraction of 7721 hepatocarcinoma cells. It was found that the enzyme activity was elevated by low concentrations of some purine nucleotides, but the activating effects were decreased when the concentrations of the nucleotides were higher. The optimal concentrations of GTP, GTPgamma[S], GDP and ATP for maximal activation were 0.1 mM, 5 microM, 1 mM and 1 mM respectively. The activation caused by 1 mM ADP was lower. The enzyme was not activated by 1 mM AMP, but significant activation was observed by the addition of 1 mM cAMP. The latter was mediated by protein kinase A, as a specific inhibitor of protein kinase A abolished the activation. There were synergic effects between ATP and GTP, ATP and PIP2, but not between ATP and GTPgamma[S], or PIP2 and GTPgamma[S]. The activating effects of GTP and ATP were abolished by neomycin, a PIP2 scavenger. These results suggest that phospholipase D is regulated by GTP-binding protein and the presence of PIP2 is required for the activation induced by GTP. Protein 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.
Huang
Y
Y
Key laboratory of Glycoconjugate Research, Ministry of Health and Department of Biochemistry, Shanghai Medical University, People 's Republic of China.
Zhang
X Y
XY
Liu
F
F
Chen
H L
HL
D000227:Adenine Nucleotides / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000473:pathology; D009355:Neomycin / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D009355:Neomycin