Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs
| Autor: | Vasanthi Nachiappan, Ram Rajasekharan, Hiranya S. Roychowdhury |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
chemistry.chemical_classification
Azides Photochemistry Substrate (chemistry) 1-Acylglycerophosphocholine O-Acyltransferase Affinity Labels Photoaffinity Labels Biochemistry Molecular Weight chemistry.chemical_compound Acyl-CoA Enzyme Membrane Biosynthesis chemistry Membrane protein Microsomes Microsome Acyl Coenzyme A Soybeans |
| Zdroj: | European journal of biochemistry. 220(3) |
| ISSN: | 0014-2956 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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