Isolation of peroxisomal enoyl-CoA hydratase in rainbow trout and immunochemical identification with the bifunctional enzyme

Abstract

Peroxisomes are the sites for β-oxidation of long-chain fatty acids. The peroxisomal bifunctional enzyme (PBE) enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase catalyzes the second and third reactions of the β-oxidation system. Originally termed PPA-80 for peroxisome-proliferation associated 80,000 MW polypeptide, PBE levels are monitored to measure peroxisome proliferation in rodents and other species. The quantity of a 79,000 MW polypeptide in the light mitochondrial fraction of the liver, as analyzed by SDS-PAGE, increases when rainbow trout are exposed to peroxisome proliferating agents. This correlates with increases in acyl-CoA oxidase activity and peroxisome volume density. In the present study, peroxisomal enoyl-CoA hydratase was purified from trout liver and analyzed by immunoblotting with anti-PBE. A positive reaction with the 79,000 MW polypeptide band was observed providing strong evidence that this is the bifunctional enzyme.