PURIFICATION AND CHARACTERIZATION OF GLYCEROPHOSPHATE ACYLTRANSFERASEFROM RAT-LIVER MITOCHONDRIA

Glycerophosphate acyltransferase (GAT) catalyzes the conversion of sn- glycerol 3-phosphate to lysophospha tidic acid (LPA), the first and co mmitted step of triacyl glycerol and phospholipid synthesis. In spite of the important regulatory roles GAT may play in this biosynthetic pa thway, little information is available on the structure, biochemical p roperties, and regulation of GAT from eukaryotic cells. We solubilized GAT from rat Liver mitochondrial membranes and purified it to an appa rent homogeneity by hydroxylapatite chromatography, preparative isoele ctric focusing, and gel filtration. The enzyme is composed of a single polypeptide of 85 kDa as determined by sodium dodecyl sulfate-polyacr ylamide gel electrophoresis and gel filtration chromatography of the n ative protein. The GAT activity was completely lost during the purific ation procedure and required addition of exogenous phospholipids for i ts reconstitution. Since a high phospholipid to detergent ratio was ne eded for full reactivation, it is concluded that GAT requires ''lipid boundary'' for reconstitution. The ability of different phospholipids to reactivate GAT decreased in the following order: phosphatidylglycer ol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), asol ectin, phosphatidylinositol (PI), phosphatidylserine (PG), and cardiol ipin. 1,2-Dioleoyl derivatives of PG; and PE were more effective in re constituting the GAT activity than corresponding dipalmitoyl derivativ es. The GAT activation was further increased by using a combination of PG and PE or PG and PC. Regardless of the phospholipid used for recon stitution, palmitoyl-CoA was the best acyl donor and LPA was the only reaction product.