Bovine brain microsomal CDP-diacylglycerol synthetase: solubilization and properties.
CDP-diacylglycerol(DAG) synthetase (EC 2.7.7.41) has been solubilized from bovine brain microsomes by the detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate). Optimal solubilization with 1.5% CHAPS yielded 55-60% of the synthetase activity. The effect of CHAPS on the enzyme was biphasic inhibiting at 0.3% and giving maximal activity at 0.5% (the concentration used for all assays). The solubilized, but not the microsomal enzyme is activated by phosphatidylcholine (PC) and strongly inhibited by cardiolipin and lysoPC. Strong inhibition by N-ethylmaleimide, 5,5'-dithio-bis (2-nitrobenzoic acid) and p-chloromercuribenzoate supported a sulfhydryl requirement for the enzyme. Phosphatidic acid (PA) from egg lecithin and 1-stearoyl,2-arachidonoyl PA were preferred substrates for the microsomal synthetase. Solubilized synthetase showed selectivity for the latter PA which is consistent with this enzyme functioning to help form the preponderant 1-stearoyl,2-arachidonoyl species of phosphatidylinositol. Further attempts to purify the synthetase were unsuccessful. All findings suggested the enzyme exists as an unstable complex.
Lin
C H
CH
Department of Biochemistry, University of Western Ontario, Canada.
Lin
J
J
Strickland
K P
KP
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002793:Cholic Acids; D004789:Enzyme Activation; D008861:Microsomes / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity
D002793:Cholic Acids; D010743:Phospholipids; D009713:Nucleotidyltransferases; C022348:phosphatidate cytidylyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate