Some studies on the metabolism of phospholipids in plasma membranes from rat liver

Abstract

1. 1. Rat liver plasma membranes were isolated by rate-isopycnic zonal centrifugation. A method is described for the size 14 zonal rotor. The isolated membranes had an isopycnic banding density of 41.2% sucrose (w/w). On the basis of studies with eleven marker enzymes and electron microscopy, at least 90% of the fraction consisted of plasma membranes. The electron micrographs showed a predominantly vesicular appearance with few junctional complexes.

2. 2. Plasma membranes exhibited virtually no CDP-choline: 1, 2-diacyl-sn-glycerol cholinephosphotransferase activity and thus appear incapable of significant in vitro synthesis of phosphatidylcholine by the cytidine nucleotide pathway.

3. 3. Phosphatidylglycerol was the major product of sn-glycerol-3-phosphate esterification by plasma membranes in the presence of CDP-diglyceride at pH 7.5. The product was identified by thin-layer chromatography using pure phosphatidylglycerol as a reference compound, and by paper chromatography of the products obtained by hydrolysis with phospholipase D.

4. 4. Incubation of plasma membranes with labeled exogenous phospholipids in the presence of Ca2+ resulted in the formation of fatty acids and monoacylphosphoglycerides. Analyses of the reaction products indicated that the phospholipase A mainly attacks the 2-position of the phosphoglyceride molecule. Plasma membrane phospholipase A also acted on labeled endogenous phosphatidylethanolamine in the presence of Ca2+.

5. 5. A partial characterization of the phospholipase indicated that it required Ca2+ for optimal activity, was insensitive to N-ethylmaleimide and deoxycholate, and was relatively heat stable. Its pH optimum was 8.0. The following was the preferred order of hydrolysis: phosphatidylethanolamine > phosphatidylglycerol > > > phosphatidylcholine.

6. 6. Lysophospholipase was not detected in rat liver plasma membranes under conditions in which maximal activity of this enzyme was observed in rat liver 100 000 × g supernatant.