Purification and Kinetic Properties of Acyl and Alkyl Dihydroxyacetone Phosphate Oxidoreductase

Abstract

Abstract The enzyme NADPH:acyl or NADPH:alkyl dihydroxyacetone phosphate oxidoreductase has been purified from Ehrlich ascites cell microsomes and guinea pig liver mitochondria. The membrane bound enzyme was extracted by the treatment of the particles with a sodium deoxycholate-KCl-NADPH solution and purified by chromatography on either a Sephadex G-200 or a Sepharose 4B column. The enzyme from ascites cell microsomes was eluted from the Sepharose column in two peaks, A and B. Peak A came out in the void volume of the column and probably represented small membrane fragments. The elution position of the Peak B enzyme suggested that it was a large molecule. However, on sucrose density gradient centrifugation the Peak B enzyme had a sedimentation coefficient of 5 S. This low sedimentation value was probably due to the high phospholipid content of the purified enzyme. The over-all purification of the enzyme from the microsomes was 5-fold. The solubilized enzyme was very unstable and could only be stabilized by adding the cosubstrate NADPH. The enzyme was shown to bind [4-3H]NADPH by Sephadex chromatography. NADPH was present during all stages of enzyme purification and storage. The purified enzyme had similar Km values toward both acyl and alkyl dihydroxyacetone phosphate. Competition experiments with the two substrates and heat denaturation studies indicate that the same enzyme reduced both acyl and alkyl dihydroxyacetone phosphate. The product NADP+, but not 1-acyl- or 1-alkyl-sn-glycerol 3-phosphate, inhibited the enzyme reaction. 1-Acyl-rac-glycerol 3-phosphate in the presence of NADP+ was not oxidized by the enzyme showing that the reaction was probably not reversible. The dephosphorylated substrates acyl and alkyl dihydroxyacetone inhibited the enzymatic reduction of acyl or alkyl dihydroxyacetone phosphate and the mode of inhibition showed that the dihydroxyacetone lipids compete with the substrates for the same active site of the enzyme. Kinetic data and the mode of product inhibition suggested that the substrates form a ternary complex with the enzyme.