Purification and characterization of thiol-reagent-sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus.
Glycerol-3-phosphate acyltransferase (GPAT), responsible for the first committed, rate-limiting, step of glycerolipid synthesis, was purified to homogeneity from the membrane fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the membrane fraction by pretreatment with 0.05% Triton X-100 and treatment of the resulting pellet with 0.3% Triton X-100. The enzyme was subsequently purified by column chromatography on heparin-Sepharose, Yellow 86 agarose, a second heparin-Sepharose column, Superdex-200 and hydroxylapatite Bio-Gel. Enzyme activity was finally enriched 1308-fold over that of the starting membrane fraction. SDS/PAGE of the purified fraction revealed a single band with a molecular mass of 45 kDa. Native PAGE showed a major band that corresponded to GPAT activity. Enzyme activity was inhibited by thiol reagents, suggesting that it originated from microsomes rather than mitochondria. Purified GPAT depended on exogenous oleoyl-CoA and sn-glycerol-3-phosphate, with the highest activity at approx. 50 and 250 microM, respectively, and preferred oleoyl-CoA 5.4-fold over palmitoyl-CoA as an acyl donor. Anionic phospholipids, such as phosphatidic acid and phosphatidylserine, were absolutely required for activity of the purified enzyme, and their ability to activate GPAT was influenced by the purity of the GPAT preparation. Bivalent cations, such as Mg(2+) and Ca(2+), inhibited purified GPAT activity, whereas 5 mM Mn(2+) elevated activity approx. 2-fold. These results provide new insights into the molecular characterization of microsomal GPAT, which has not been well characterized compared with mitochondrial and plastidic GPAT.
Mishra
S
S
Applied Microbiology Department, National Institute of Bioscience & Human Technology, Tsukuba, Ibaraki 305-8566, Japan.
Kamisaka
Y
Y
D002462:Cell Membrane / Q000201:enzymology; D002853:Chromatography, Liquid; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D020094:Mortierella / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
D004228:Dithionitrobenzoic Acid; D005992:Glycerol-3-Phosphate O-Acyltransferase; D004229:Dithiothreitol