Aw. Tumaney et R. Rajasekharan, Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons, BBA-MOL C B, 1439(1), 1999, pp. 47-56
Citations number
37
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS
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.