Use of photoreactive substrates for characterization of lysophosphatidate acyltransferases from developing soybean cotyledons.
Photoreactive lipid analogs, namely, 1-acyl-2-(12-azidooleoyl)glycero-3-phosphocholine (N3-PC) and 1-acyl-2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-PE) have been synthesized as previously described [R. Rajasekharan and J. D. Kemp (1994) J. Lipid Res. 35, 45-51]. Azidophosphatidic acid was produced by hydrolyzing N3-PC with phospholipase D. All of the lysophospholipid analogs, 2-(12-azidooleoyl)glycero-3-phosphate (N3-LPA), 2-(12-azidooleoyl)glycero-3-phosphocholine (N3-LPC), and 2-(12-azidooleoyl)glycero-3-phosphoethanolamine (N3-LPE), were produced from appropriate azidophospholipids by lipase treatment. The photoactive lysophospholipid analogs were recognized as substrates by acyltransferases in the dark and as irreversible inhibitors after photolysis with uv light. The photoinactivation of acyltransferases by azidolysophospholipids was protected by the addition of natural lysophospholipids. Incubation of developing soybean microsomal membranes with N3-LPA followed by photolysis resulted in 69% inhibition of lysophosphatidic acid (LPA) acyltransferase and also had significant inhibitory effects on lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) acyltransferases, indicating that the LPA analog interacts with all the lysophospholipid acyltransferases. When the membranes were photolyzed with N3-LPC or N3-LPE and assayed, the membranes showed approximately 50% inactivation of LPC and LPE acyltransferase activities, whereas LPA acyltransferase was unaffected, suggesting that a single enzyme might acylate both LPC and LPE. The recognition of these photoreactive lipid analogs by acyltransferases will facilitate the identification and purification of these membrane-bound enzymes.
Rajasekharan
R
R
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces 88003.
Nachiappan
V
V
D000217:Acyltransferases / Q000378:metabolism*; D003624:Darkness; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008027:Light; D008861:Microsomes / Q000201:enzymology*; D010714:Phosphatidylethanolamines / Q000138:chemical synthesis* / Q000378:metabolism*; D010743:Phospholipids / Q000138:chemical synthesis* / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D010714:Phosphatidylethanolamines; D010743:Phospholipids; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase