Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs.
Synthesis of 32P-labeled 12-azidooleoyl-CoA and 125I-labeled 12-[(azidosalicyl)amino]dodecanoyl-CoA (ASD-CoA) was achieved. The synthesized radioactive, photoreactive reagents were tested as photoaffinity labels for acyl-CoA:lysophosphatidylcholine acyltransferase from the microsomal membranes of developing soybean cotyledons. When a mixture of microsomal membranes and the azidooleoyl-CoA or ASD-CoA were incubated in the dark, the analogs were recognized as substrate and competitive inhibitor, respectively. The enzyme preferentially utilizes unsaturated acyl-CoAs rather than saturated acyl-CoAs. Incubation of microsomal membranes with acyl-CoA analogs and immediately followed by photolysis resulted in an irreversible inhibition of lysophosphatidylcholine acyltransferase activity. Analysis of photolyzed microsomal membranes by SDS/PAGE and autoradiography revealed that azidooleoyl-CoA preferentially labeled eight acyl-CoA binding proteins, but ASD-CoA labeled only three polypeptides with molecular masses of 110, 90 and 32 kDa that are commonly labeled by both the analogs. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme against photoinactivation by azidooleoyl-CoA and ASD-CoA, respectively. The protection was profound in 110-kDa polypeptide indicating that this protein could be lysophosphatidylcholine acyltransferase. These results demonstrate that the photoaffinity of acyl-CoA analogs makes them potential probes to identify and characterize lipid biosynthetic enzymes.
Rajasekharan
R
R
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces 88003.
Nachiappan
V
V
Roychowdhury
H S
HS
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000737:chemistry*; D000345:Affinity Labels; D001386:Azides / Q000737:chemistry; D008861:Microsomes / Q000737:chemistry*; D008970:Molecular Weight; D010777:Photochemistry; D013025:Glycine max
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D001386:Azides; D008243:1-Acylglycerophosphocholine O-Acyltransferase