Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes.
G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.
Silk
S T
ST
Department of Biochemistry, New York College of Osteopathic Medicine, Old Westbury 11568.
Clejan
S
S
Witkom
K
K
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000097:blood* / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D012265:Ribonucleotides / Q000494:pharmacology; D015398:Signal Transduction; D013873:Thionucleotides / Q000494:pharmacology
D001095:Arachidonic Acids; D012265:Ribonucleotides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D004492:Edetic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium