Enzymic Synthesis of O-Alkyl Bonds in Glycerolipids

Abstract

We have found that O-alkyl bonds in glycerolipids are synthesized by microsomal enzymes of preputial gland tumors when dihydroxyacetone-P or glyceraldehyde-3-P and 1-14C long chain fatty alcohols are used as substrates; ATP, coenzyme A, and Mg++ are essential cofactors. The O-alkyl moieties formed from the 1-14C- and 9,10-3H-labeled long chain fatty alcohols show that the fatty alcohol chains are directly incorporated. Dihydroxyacetone-P is an obligate source of the glycerol portion of glyceryl ethers, since only the dihydroxyacetone-P served as a precursor when 1-hydroxy-3-chloro-2-propanone phosphate was used as a specific inhibitor of triose-P isomerase, which is present in the microsomal preparation. Kinetic data on the following parameters were obtained: time, CoA, Mg++, ATP, protein, pH, and inhibitors (phosphate and 1-hydroxy-3-chloro-2propanone phosphate). We documented the chromatographic behavior of the intermediates (O-alkyldihydroxyacetone and O-alkyldihydroxyacetone-P) and identified the O-alkyl linkage by thin layer and gas-liquid chromatography of the isopropylidene derivatives of the O-alkylglycerols formed by LiAlH4 reduction. Only the 1 isomeric O-alkylglycerols could be isolated after LiAlH4 reduction. Although other nucleotides (ADP and ITP) substituted for ATP in this system, glutathione or cysteine could not replace the CoA requirement. Metals other than Mg++ did not provide optimal conditions, and copper ions inhibited the biosynthesis of O-alkyl bonds. To some extent, dihydroxyacetone was able to replace the dihydroxyacetone-P requirement, but it is possible that phosphorylation occurred in the system. The enzymic synthesis of the O-alkyl linkage is localized in microsomes and appears to decrease as the tumor ages. The preputial gland tumors contain significant quantities of Oalkyl- and O-alk-1-enyl-linked lipid classes, but the enzyme system described was unable to make O-alk-1-enyl bonds under our experimental conditions. However, the similar composition of the O-alkyl and O-alk-1-enyl moieties (16:0, 18:0, and 18:1) suggests that interconversions between these two lipid classes or their precursors may occur.