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

Ajay, Tumaney W and Ram, Rajasekharan (1999) Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons. In: Biochimica et Biophysica Acta, 1439 (1). pp. 47-56.

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Abstract

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-[(4-azidosalicyl)-12-amino]dodecanoyl-sn-glycerol-3-phosphocholine (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.

Item Type:	Journal Article
Publication:	Biochimica et Biophysica Acta
Publisher:	Elsevier B.V
Additional Information:	copyright of this article belongs to Elsevier B.V
Keywords:	Enzymology
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	18 Oct 2007
Last Modified:	19 Sep 2010 04:40
URI:	http://eprints.iisc.ac.in/id/eprint/12138

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