Occurrence and characterization of a dehydratase enzyme in the leek icosanoyl-CoA synthase complex.
The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.
Lessire
R
R
I.B.C.-CNRS, Université de Bordeaux II, France.
Bessoule
J J
JJ
Cook
L
L
Cinti
D L
DL
Cassagne
C
C
D000490:Allium / Q000201:enzymology*; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006836:Hydro-Lyases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology; D051381:Rats; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000906:Antibodies; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C065185:D-3-hydroxyacyl CoA dehydratase; D006836:Hydro-Lyases; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase