ETHER LIPID-SYNTHESIS - PURIFICATION AND IDENTIFICATION OF ALKYL DIHYDROXYACETONE PHOSPHATE SYNTHASE FROM GUINEA-PIG LIVER

Alkyl-dihydroxyacetone phosphate synthase, the second enzyme involved in ether phospholipid biosynthesis from dihydroxyacetone phosphate and responsible for glycero-ether bond formation, has been purified from guinea-pig liver. Alkyl-dihydroxyacetone phosphate synthase was solubi lized from a membrane fraction prepared from an enriched peroxisome fr action with Triton X-100 and potasssium chloride. The solubilized enzy me was further purified by chromatography on QAE-Sephadex, Matrex Red, Phosphocellulose and Concanavalin A. Upon sodium dodecyl sulfate-poly acrylamide gel electrophoresis alkyl-dihydroxyacetone phosphate syntha se appears as a 65 kDa band. Chromatofocusing revealed an isoelectric point of pH 5.9 for the enzyme. The pH optimum of alkyl-dihydroxyaceto ne phosphate synthase was found to be between pH 7 and 8 in a 50 mM po tassium phosphate buffer. The specific activity of the enzyme was esti mated to be at least 350 nmol . min-1 . mg corresponding to a purifica tion of at least 13 000-fold.