The Presence of a New 3‐Oxoacyl‐CoA Thiolase in Rat Liver Peroxisomes

Abstract

A novel 3-oxoacyl-CoA thiolase was found in rat liver. This thiolase, mitochondrial general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase were purified from the rat liver after the induction of these activities by the administration to rats of di(2-ethylhexyl)phthalate, which enhanced the peroxisomal P-oxidation activity. The new 3-oxoacyl-CoA thiolase was distinguished from mitochondrial and cytoplasmic thiolases by the following : DEAE-cellulose chromatography, phosphocellulose chromatography, immunochemical titration, and substrate specificity. Subcellular fractionation of liver and sucrose-density-gradient centrifugation of the light mitochondrial fraction revealed that the new thiolase was mainly in peroxisomes.Mammalian liver has three types of thiolases which differ in the intracellular localization [I 1. Cytoplasmic acetoacetyl-CoA thiolase plays a role in steroid biosynthesis. Mitochondria have two different thiolases. One of them is specific for acetoacetyl-CoA and is supposed to have a role for ketone body metabolism. The other, a general 3-oxoacyl-CoA thiolase, is involved in P-oxidation of fatty acid. Recently, it has been shown that rat liver peroxisomes have a new P-oxidation system and the activity of this system is markedly increased when a rat is fed a diet containing various hypolipidemic agents [2,3] or di(2-ethylhexy1)-phthalate [4]. Hepatic thiolase activities increased in the di(2-ethy1hexyl)phthalate-treated rats [5]. The increments of the activities for substrates with longer carbon-chain length were greater than that for acetoacetyl-CoA. The subcellular fractionation study suggests that peroxisomes have an unique thiolase different from cytoplasmic and mitochondrial thiolases [5].The present paper describes the purification of a new 3-oxoacyl-CoA thiolase and its location in peroxisomes. MATERIALS AND METHODS MaterialsAcetoacetyl-CoA was prepared by the reaction of diketene with CoA [6]. 2-Octenoyl-CoA was syn-

Abnormal Lipid Metabolism in Cystathionine β-Synthase-deficient Mice, an Animal Model for Hyperhomocysteinemia

“…The protein content of the supernatant was determined with the Bio-Rad protein assay. Thiolase activity was measured as described previously (19). Briefly, the reaction was initiated by adding tissue samples to a reaction mixture containing 50 M 3-oxooctanoyl-CoA, 0.15 mM CoA, 25 mM MgCl 2 , 50 mM KCl, and 100 mM Tris-HCl (pH 8.0).…”

Immunological assay of the mitochondrial acetyl‐CoA acetyltransferase in crude liver homogenate

“…The mAAT was proved to be immunologically distinct from the mitochondrial acetyl-CoA acyltransferase, from the cytosolic acetyl-CoA acetyltransferase [2] and from the peroxisomal acetyl-CoA acyltransferase [3]. Therefore rocket immunoelectrophoresis can be employed to quantitate the amount of mAAT directly in liver homogenates with purified mAAT as a standard.…”

Genetic heterogeneity in patients with a disorder of peroxisomal β‐oxidation: A complementation study based on pristanic acid β‐oxidation suggesting different enzyme defects

“…It is clear that there are two acyl-CoA oxidases with specificity for straight-chain and branched-chain fatty acyl-CoA esters (Vanhove et al 1993). Until recently it was believed that the subsequent steps are catalysed by one bifunctional protein and peroxisomal thiolase (Miyazawa et al 1980), but this view is no longer tenable (see Novikov et al 1994).…”

“…It is clear that there are two acyl-CoA oxidases with specificity for straight-chain and branched-chain fatty acyl-CoA esters (Vanhove et al 1993). Until recently it was believed that the subsequent steps are catalysed by one bifunctional protein and peroxisomal thiolase (Miyazawa et al 1980), but this view is no longer tenable (see Novikov et al 1994).We have recently found that the bifunctional protein and thiolase as characterized by Hashimoto and coworkers are not involved in pristanic acid β-oxidation. In collaboration with Seedorf and coworkers we have shown that the thiolase encoded by the sterol carrier protein X (SCPx) gene (Seedorf et al 1994) contains 3-ketopristanoyl-CoA thiolase activity, whereas the classical thiolase lacks such activity (Wanders et al 1996).…”