Peroxisomal β-ketothiolase

Abstract

β-Ketothiolase was found in rat liver peroxisomes that were isolated by sucrose density gradient centrifugation. The presence of dithiothreitol was essential for measuring the peroxisomal thiolase, but dithiothreitol had little effect upon the thiolase activity in the mitochondria or cytosol. Dithiothreitol was not used during the isolation procedures. Acetoacetyl CoA and β-ketolauryl CoA, which was synthesized chemically, were used as substrates. The peroxisomal thiolase was active with β-ketolauryl CoA but had almost no activity with acetoacetyl CoA as the substrate. Thiolase activities in the mitochondrial and cytosolic fractions utilized both long- and short-chain substrates. The thiolases in the various fractions bound to and were purified by chromatography on calcium phosphate gel cellulose columns. The peroxisomal thiolase did not bind to phosphocellulose, whereas the mitochondrial and cytosolic activities could be chromatographed on phosphocellulose. Peroxisomal β-ketolauryl CoA thiolase activity was inhibited about 20% by 25 m m Mg 2+, and more activity was measured in a phosphate than in a Tris buffer. In control rat livers, the total activity of β-ketolauryl CoA thiolase was 3.4 μmol/min per gram of liver in the peroxisomes and 4.9 μmol/min per gram of liver in the mitochondria, which indicates that peroxisomes contain the capacity for 40% of the total thiolase activity associated with β-oxidation systems. In addition, 6.3 μmol/min per gram of liver of β-ketolauryl CoA thiolase was found in the soluble fraction and chromatographed differently from the peroxisomal enzyme on phosphocellulose. Clofibrate in the rat diet for 6 or 21 days resulted in about a 15-fold increase in peroxisomal thiolase when assayed with β-ketolauryl CoA and somewhat less of an increase in the other fractions.