Partial purification and properties of glycerophosphate acyltransferase from rat liver. Formation of 1-acylglycerol 3-phosphate from sn-glycerol 3-phosphate and palmityl coenzyme A.

Abstract

1 Glycerophosphate acyltransferase was partially purified from rat-liver microsomes which were resolved with a nonionic detergent, Triton X-100, in glycine buffer pH 8.6. The purification procedure involves molecular-sieve chromatography and sucrose density gradient centrifugation. 2 The partially purified enzyme requires Ca2+ for its activity. Divalent cations, such as Mg2+, Mn2+ and Co2+, are able to substitute for Ca2+ with varying degrees of effectiveness. Ca2+ in higher concentrations is inhibitory, but this inhibition is abolished by the addition of small amounts of phospholipids. Moreover, phospholipids stimulate the enzyme activity at all concentrations of Ca2+ tested. The pH optimum of the enzyme is broad, extending over a pH range from 6.6 to 9.0. 3 The apparent Michaelis constant of the partially purified enzyme for sn-glycerol 3-phosphate is 0.2 mM, which approximates the value found with unresolved microsomes (0.5 mM). 4 Among the acyl donors examined, palmityl-CoA is utilized most efficiently by the partially purified enzyme. Unsaturated fatty acyl-CoA thioesters, such as oleyl-CoA, linoleyl-CoA and arachidonyl-CoA, are poor substrates. 5 The partially purified enzyme catalyzes the formation of monoacylglycerol 3-phosphate from sn-glycerol 3-phosphate and palmityl-CoA, esterifying preferentially position 1 of the glycerol moiety. Little phosphatidic acid is produced even in the presence of both palmityl-CoA and linoleyl-CoA. Thus the acyltransferase preparation is free of the enzyme responsible for acylation of 1-acylglycerol 3-phosphate. 6 The results presented indicate that phosphatidic acid synthesis in mammalian liver proceeds through a sequential acylation of sn-glycerol 3-phosphate mediated by two distinct acyltransferases. The data also show that acylation of sn-glycerol 3-phosphate to monoacylglycerol 3-phosphate occurs in a non-random manner, thus contributing to the asymmetric distribution of fatty acids in glycerolipids.