Biosynthesis of an O-alkyl analogue of phosphatidic acid and O-alkylglycerols via O-alkyl ketone intermediates by microsomal enzymes of Ehrlich ascites tumor.

Microsomal enzymes from Ehrlich ascites tumors synthesized 0-alkyl glyceryl ethers from hexadecanol and dihydroxyacetone phosphate in the presence of ATP, CoA, and Mg++. Glyceryl ether synthesis occurred via the formation of 0-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 I-0-alkylglycerophosphate and I-O-alkylglycerol. 1-0-Alkylglycerophosphate was acylated by endogenous fatty acids in the Ehrlich ascites microsomes to form I-0-alkyl-Z-acyl glycerophosphate. In contrast, no acylation of I-0-alkylglycerol occurred. The formation of an 0-alkyl analogue of phosphatidic acid suggests that Oalkyl glycerolipids are synthesized by the well known cytidine pathway. The 0-alkylglycerols were identified as isopropylidene derivatives and as 0-alkyl glycolic aldehydes after periodate oxidation. The biosynthesis of 0-alkyl ether bonds was measured as a function of time, pH, and the concentrations of protein, 1-14C-hexadecanol, dihydroxyacetone phosphate, Mgf+, 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 lhydroxy-3-chloro-Z-propanone phosphate. 0-Alkyldihydroxyacetone was identified by the following methods: periodate oxidation to 0-alkyl glycolic acid; reduction to 0-alkyl glyceryl ethers by LiAlH4, hydrogenation, NaBH4, and NADPH (enzymatically); reduction by LiAlH4 of the 0-alkyl glycolic acid (derived from 0-alkyl dihydroxyacetone) to 0-alkyl ethylene glycol; and identification of the