Purification and characterization of glycerophosphate acyltransferase from rat liver mitochondria.
Glycerophosphate acyltransferase (GAT) catalyzes the conversion of sn-glycerol 3-phosphate to lysophosphatidic acid (LPA), the first and committed step of triacylglycerol and phospholipid synthesis. In spite of the important regulatory roles GAT may play in this biosynthetic pathway, little information is available on the structure, biochemical properties, and regulation of GAT from eukaryotic cells. We solubilized GAT from rat liver mitochondrial membranes and purified it to an apparent homogeneity by hydroxylapatite chromatography, preparative isoelectric focusing, and gel filtration. The enzyme is composed of a single polypeptide of 85 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography of the native protein. The GAT activity was completely lost during the purification procedure and required addition of exogenous phospholipids for its reconstitution. Since a high phospholipid to detergent ratio was needed 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: phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), asolectin, phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin. 1,2-Dioleoyl derivatives of PG and PE were more effective in reconstituting the GAT activity than corresponding dipalmitoyl derivatives. The GAT activation was further increased by using a combination of PG and PE or PG and PC. Regardless of the phospholipid used for reconstitution, palmitoyl-CoA was the best acyl donor and LPA was the only reaction product.
Vancura
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A
Department of Biological Sciences, St. John's University, Jamaica, New York 11439.
Haldar
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D
D000818:Animals; D004789:Enzyme Activation; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007526:Isoelectric Point; D008297:Male; D008565:Membrane Proteins / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013379:Substrate Specificity
D008565:Membrane Proteins; D010743:Phospholipids; D005992:Glycerol-3-Phosphate O-Acyltransferase