Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-[(4-az idosalicyl)-12-amino]dodecanoyl-sn-glycerol-3-phosphocholine (azido-LPC) wa s synthesized. Fast atom bombardment mass spectrometry was employed to conf irm the structures of azido-LPC and its intermediates. Azido-LPC was used t o label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a s ubstrate for the target acyltransferases and phospholipases in the dark. Wh en the microsomal membranes were incubated with the acyl acceptor analog an d immediately photolyzed, LPC acyltransferase was irreversibly inhibited. P hotoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with I-125-labeled azido-LPC a nd analyzed by SDS-PAGE followed by autoradiography. These revealed that th e analog preferentially labeled 54- and 114-kDa polypeptides. Substrate pro tected the labeling of both the polypeptides. In our earlier report, the sa me polypeptides were also labeled with photoreactive acyl-CoA analogs, sugg esting that these polypeptides could be putative LPC acyltransferase(s). Th ese results demonstrated that the photoreactive phospholipid analog could b e a powerful tool to label acyltransferases involved in lipid biosynthesis. (C) 1999 Elsevier Science B.V. All rights reserved.