Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Abstract

Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacylCoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments.The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

Cloning and expression pattern of peroxisomal β-oxidation genes palmitoyl-CoA oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase in mussel Mytilus galloprovincialis and thicklip grey mullet Chelon labrosus

“…Both rodent enzymes have the same substrate specificities (Antonenkov et al, 1999;Chevillard et al, 2004), but they are differently regulated: thiolase A is constitutively expressed whereas the expression of thiolase B is induced by peroxisome proliferators (Antonenkov et al, 2000;Hijikata et al, 1990). Fish seem to show the same enzymatic content as humans or chicken, and thus thio and scpx orthologs have been found in the 5 teleost genomes available (Table 3).…”

The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles

“…The thiolytic cleavage activity of peroxisomal fractions toward acetoacetyl-CoA was determined at 306 nm (⑀: 3,600 M Ϫ1 /cm). In normal rat liver peroxisomes, the bulk of this activity is determined by soluble matrix 3-oxoacyl-CoA thiolase (4,6). Units of enzyme activity are expressed as micromoles of substrate consumed or product formed per minute.…”

Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells

“…Animal care approval was granted by the local institutional ethics committee. The rabbit polyclonal antibodies against (His) 6 -Pex13p, (His) 6 -Pex14p, (His) 6 -Pex19p, peroxisomal thiolase and GST were raised and employed as described elsewhere [31,44,45]. The rabbit anti-FTase antibody was obtained from Calbiochem, and the anti-farnesyl and secondary antibodies were purchased from Sigma.…”