RESUMO
Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20:0-CoA and (E) 2,3-20:1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 microM 18:1-CoA and 30 microM malonyl-CoA, and that 18:0-CoA and 18:1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 microM Triton X-100 and 25 microg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Aciltransferases/química , Aciltransferases/isolamento & purificação , Brassica/enzimologia , Hidroliases/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila S-Maloniltransferase , Cromatografia em Agarose , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hidroliases/metabolismo , Cinética , Malonil Coenzima A/farmacologia , Octoxinol/farmacologia , Sefarose/química , Fatores de TempoRESUMO
Polyclonal antibodies have been raised against the acyl-CoA elongase purified from leek epidermal cells. The antibodies recognize the fractions containing the elongating activity after DEAE or Ultrogel chromatography and their response with the other fractions is very low. The immune complex is immunoprecipitable with Protein A-Sepharose. 1% of the solubilized proteins from leek epidermis microsomes are immunoprecipitated. The immunoprecipitate contains an elongating activity which is 86 +/- 20-times that of the unbound fraction.
