The soluble TAG biosynthesizing enzymes were purified by native PAGE and the enzyme activities localized on the gel. TAG is synthesized from LPA through a sequential acylation process involving lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase. The 10S multienzyme complex also contains acyl carrier protein and acyl-acyl carrier protein synthetase. This is the first report on the identification and characterization of a soluble 10S TAG biosynthesizing multienzyme complex. Identification of acyltransferases involved in lipid biosynthesis using photoreactive substrate analogs.
Enzymes involved in lipid biosynthesis are mostly membrane-bound.
Attempts to solubilize and purify these enzymes have been largely unsuccessful. An alternative approach was used, which involves covalent labeling of proteins with site directed ligand bearing photoreactive group. Upon photolysis, the photoprobe was incorporated selectively into a 29-kDa polypeptide in the microsomal membranes. These studies indicate that the photoaffinity reagents could be used as a powerful tool to identify the membrane-bound enzymes. Phosphatidyl choline is one of the major phospholipid constituents of cellular membranes.