Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds.
Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.
D000217:Acyltransferases / Q000302:isolation & purification*; D000818:Animals; D002099:Cacao / Q000201:enzymology*; D002850:Chromatography, Gel; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010945:Plants, Edible / Q000201:enzymology*; D011817:Rabbits; D011863:Radioimmunoassay; D012639:Seeds / Q000032:analysis; D012995:Solubility
D046911:Macromolecular Substances; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase