A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase.
1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate.
Rajasekharan
R
R
Department of Microbiology, University of Illinois, Urbana 61801.
Ray
T K
TK
Cronan
J E
JE
D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; D000217:Acyltransferases / Q000032:analysis*; D000469:Alkaline Phosphatase; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D010761:Phosphorus Radioisotopes; D012995:Solubility
D029968:Escherichia coli Proteins; D010761:Phosphorus Radioisotopes; D000217:Acyltransferases; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C495091:plsC protein, E coli; D000469:Alkaline Phosphatase