Purification of dihydroxyacetone phosphate acyltransferase from guinea pig liver peroxisomes.
Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone phosphate acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have a M(r) of 69,000. This was verified by further purifying the enzyme by chromato-focusing, when a single 69-kDa band was observed on SDS-PAGE. The M(r) of dihydroxyacetone phosphate acyltransferase determined by gel filtration is 90 kDa. The Vmax of the purified enzyme was approximately 4 mumol acyldihydroxyacetone phosphate (acylDHAP) formed per minute per milligram protein and the Km(DHAP) is approximately 70 microM when assayed at saturating concentrations of palmitoyl-CoA. Free coenzyme A inhibits the acyltransferase reaction with an inhibition constant (Ki) of approximately 0.76 mM. To date, this is the most highly purified DHAP acyltransferase (> 3200-fold) of mammalian origin.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
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D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D009994:Osmolar Concentration; D014461:Ultracentrifugation
D006882:Hydroxyapatites; D017886:Durapatite; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase