Studies on Acyl Coenzyme-A: Dihydroxyacetone Phosphate Acyltransferase.

Abstract

Subcellular fractionation studies in guinea pig and rat liver indicated that acyl CoA: dihydroxyacetone phosphate (DHAP) acyltransferase (EC.2.3.1.42) was enriched in the light mitochondrial (L) fraction, which contains mainly lysosomes and peroxisomes. After separation of lysosomes from peroxisomes in rat liver it was shown that the enzyme is localized in the latter fraction. The properties of DHAP acyltransferase from the guinea pig liver "L" fraction were studied in detail and compared with those of glycerol-3-P acyltransferase present in the same fraction. The pH optimum of membrane-bound DHAP acyltransferase in the absence of detergent was broad with a maximum at pH 5.5 and 20% maximal activity at pH 9.0, while with detergent it was sharp with a maximum of 7.4. The membrane-bound enzyme was fairly resistant to heat and trypsin treament and was not inhibited by high concentrations of N-ethyl maleimide. The properties of glycerol-3-P acyltransferase with respect to pH optimum, stability towards different detergents, heat, trypsin, and N-ethyl maleimide differed from those found for DHAP acyltransferase, indicating that these acyltransferases in the "L" fraction are not the same enzyme. Part of the DHAP acyltransferase activity is latent, as indicated by the stimulation of enzyme activity by detergents, by osmotic shock, and by sonication. Kinetic studies of the membrane-bound DHAP acyltransferase using varying DHAP concentrations showed a biphasic reciprocal plot at both pH 5.5 and 7.4. DHAP acyltransferase was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The liver peroxisomal DHAP acyltransferase was solubilized by treating with a solution of 1M KCl containing sodium cholate (0.2%). The enzyme was then purified by ammonium sulfate fractionation and Sepharose 6B gel filtration. Overall purification was 80-100 fold from peroxisomes with a 25-30 percent yield. On sodium dodecyl sulfate polyacrylamide gel electrophoresis the partially purified enzyme preparation showed two major protein b and s (Mr = 50,000 and 44,000) and two minor b and s (Mr = 110,000 and 33,000). By Sepharose-6B chromatography the enzyme's molecular weight was estimated to be approximately 200,000. The activity of the partially purified enzyme was stimulated by Asolectin (a soybean phospholipid preparation), by peroxisomal total lipid extract, and to a smaller extent, by phosphatidyl choline and phosphatidyl inositol. The enzyme was inhibited by several different thiol reagents and by Cu('++) and Cd('++). Dithiothreitol and Asolectin protected the enzyme from denaturation on storage. Kinetic studies with varying concentrations of DHAP showed a biphasic reciprocal plot (K(,m) 0.1 mM and 0.5 mM at pH 7.4) in the absence of Asolectin and a linear reciprocal plot (K(,m) 0.1 mM) in the presence of Asolectin. In both instances the V(,max) was 333 nmol/min/mg protein. The enzyme catalyzed an exchange of acyl groups betwen DHAP and palmitoyl DHAP in the presence of CoA, and also catalyzed the formation of acyl CoA from CoA and acyl DHAP. The partially purified enzyme preparation had negligible glycerol-3-P acyltransferase activity.