Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues.
Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [(14)C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway - as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil.
Manaf
A M
AM
Cardiff School of Biosciences, Cardiff University, P.O. Box 911, Cardiff CF1 3US, Wales, UK.
Harwood
J L
JL
D000214:Acyl Coenzyme A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010168:Palmitates / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D005994:Glycerophosphates; D010168:Palmitates; D005992:Glycerol-3-Phosphate O-Acyltransferase