Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver.
Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25-30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent Km values (0.1 and 0.5 mM) for dihydroxyacetone phosphate. The Vmax was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a Km = 0.1 mM and no change in Vmax. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [3H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [3H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.
D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D012995:Solubility; D013696:Temperature
D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase