Ether lipid synthesis: purification and identification of alkyl dihydroxyacetone phosphate synthase from guinea-pig liver.
Alkyl-dihydroxyacetone phosphate synthase, the second enzyme involved in ether phospholipid biosynthesis from dihydroxyacetone phosphate and responsible for glycero-ether bond formation, has been purified from guinea-pig liver. Alkyl-dihydroxyacetone phosphate synthase was solubilized from a membrane fraction prepared from an enriched peroxisome fraction with Triton X-100 and potassium chloride. The solubilized enzyme was further purified by chromatography on QAE-Sephadex, Matrex Red, Phosphocellulose and Concanavalin A. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis alkyl-dihydroxyacetone phosphate synthase appears as a 65 kDa band. Chromatofocusing revealed an isoelectric point of pH 5.9 for the enzyme. The pH optimum of alkyl-dihydroxyacetone phosphate synthase was found to be between pH 7 and 8 in a 50 mM potassium phosphate buffer. The specific activity of the enzyme was estimated to be at least 350 nmol.min-1.mg-1, corresponding to a purification of at least 13,000-fold.
Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
A W
AW
Zomer
W F
WF
de Weerd
J
J
Langeveld
H
H
van den Bosch
D000217:Acyltransferases / Q000378:metabolism; D019883:Alkyl and Aryl Transferases; D000818:Animals; D002845:Chromatography / Q000379:methods; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008830:Microbodies / Q000201:enzymology; D008970:Molecular Weight; D010955:Plasmalogens / Q000096:biosynthesis*; D013045:Species Specificity; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D010955:Plasmalogens; D004099:Dihydroxyacetone Phosphate; D014166:Transferases; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase