Studies on Phospholipid-synthesizing Enzyme Activities during the Growth of Etiolated Cucumber Cotyledons

The enzymatic incorporation of sn-glycerol 3-phosphate into lipid by extracts of cucumber (Cucumis sativus) cotyledons showed an absolute requirement for ATP (saturation 2 mM). The incorporation was stimulated 4-fold by 0.2 mM oleate. Ethyldiaminetetraacetate stimulated the incorporation at concentrations below 1 mM and inhibited at higher concentrations. Mg(2+) did not affect the reaction. Triton X-100 and Cutscum inhibited the reaction, while a third detergent, Span 80, was stimulatory. p-Mercuribenzoate was inhibitory. The enzymatic reaction has a pH optimum in the range of 8.8 to 9.6. The Michaelis constant was 112 muM for sn-glycerol 3-phosphate. The major amount of product was phosphatidic acid, the remainder was diacylglycerol, monoacylglycerol, and an unknown phospholipid.The activity profiles for two glyoxysmal enzymes, malate synthetase and catalase, were compared to the activities of four enzymes involved in phospholipid synthesis. Phosphatidylcholine and phosphatidylethanolamine synthesis paralleled the activity profiles of catalase and malate synthetase, as well as the levels of endogenous diglycerides. sn-Glycerol 3-phosphate incorporation peaked at a later stage of cucumber cotyledon growth than the glyoxysomal enzymes and seemed to be the major pathway of phosphatidic acid synthesis. Diglyceride phosphokinase activity did not reach appreciable levels during the first 11 days of cucumber cotyledon growth.