Photolabeling of soybean microsomal membrane proteins with photoreactive acyl-CoA analogs.
Eur J Biochem
; 220(3): 1013-8, 1994 Mar 15.
Article
em En
| MEDLINE
| ID: mdl-8143717
ABSTRACT
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-CoAlysophosphatidylcholine 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Acil Coenzima A
/
1-Acilglicerofosfocolina O-Aciltransferase
/
Microssomos
Idioma:
En
Revista:
Eur J Biochem
Ano de publicação:
1994
Tipo de documento:
Article