Biosynthesis of an O-Alkyl Analogue of Phosphatidic Acid and O-Alkylglycerols via O-Alkyl Ketone Intermediates by Microsomal Enzymes of Ehrlich Ascites Tumor

Abstract

Microsomal enzymes from Ehrlich ascites tumors synthesized O-alkyl glyceryl ethers from hexadecanol and dihydroxyacetone phosphate in the presence of ATP, CoA, and Mg++. Glyceryl ether synthesis occurred via the formation of O-alkyldihydroxyacetone phosphate and O-alkyldihydroxyacetone. Although NADPH inhibited the initial reaction of dihydroxyacetone phosphate with fatty alcohol, it was required at a later stage for reduction of the ketone intermediates to 1-O-alkylglycerophosphate and 1-O-alkylglycerol. 1-O-Alkylglycerophosphate was acylated by endogenous fatty acids in the Ehrlich ascites microsomes to form 1-O-alkyl-2-acyl glycerophosphate. In contrast, no acylation of 1-O-alkylglycerol occurred. The formation of an O-alkyl analogue of phosphatidic acid suggests that O-alkyl glycerolipids are synthesized by the well known cytidine pathway. The O-alkylglycerols were identified as isopropylidene derivatives and as O-alkyl glycolic aldehydes after periodate oxidation. The biosynthesis of O-alkyl ether bonds was measured as a function of time, pH, and the concentrations of protein, 1-14C-hexadecanol, dihydroxyacetone phosphate, Mg++, ATP, CoA, and NADPH. The Ehrlich ascites microsomes contained a triose phosphate isomerase, which we were unable to remove. We showed that dihydroxyacetone phosphate is the obligate precursor of glyceryl ethers in this system by inhibiting the isomerase with 1-hydroxy-3-chloro-2-propanone phosphate. O-Alkyldihydroxyacetone was identified by the following methods: periodate oxidation to O-alkyl glycolic acid; reduction to O-alkyl glyceryl ethers by LiAlH4, hydrogenation, NaBH4, and NADPH (enzymatically); reduction by LiA1H4 of the O-alkyl glycolic acid (derived from O-alkyl dihydroxyacetone) to O-alkyl ethylene glycol; and identification of the acetate derivative of the O-alkyl ethylene glycol. 1-O-Alkyl-2-acyl glycerophosphate was identified by the following methods: mild saponification to 1-O-alkylglycerophosphate; more drastic saponification to alkyl glyceryl ether; treatment by LiA1LH4 to yield alkyl glyceryl ether; hydrolysis with phospholipase A to yield 1-O-alkylglycerophosphate; and thin layer chromatography in acidic and basic solvent systems. O-Alkyldihydroxyacetone phosphate was identified by the following methods: removal of the phosphate by bacterial alkaline phosphatase to yield O-alkyldihydroxyacetone and subsequent identification of the O-alkyldihydroxyacetone; reduction by LiA1H4 to alkyl glyceryl ether; thin layer chromatography in acidic and basic solvent systems; and enzymatic reduction by NADPH to 1-O-alkylglycerophosphate. An equimolar incorporation of hexadecanol and dihydroxyacetone phosphate into the ether-containing intermediates and glyceryl ethers was shown with 14C-, 3H-, and 32P-labeled substrates. Data obtained with 1-14C, 1-3H-hexadecanol showed that no oxidation of the alcohol occurred during formation of ether bonds.