Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes.
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-oxoacyl-CoAs. 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.
Antonenkov
V D
VD
Departement Moleculaire Celbiologie Afdeling Farmacologie en Biochemie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Belgium.
Croes
K
K
Waelkens
E
E
Van Veldhoven
P P
PP
Mannaerts
G P
GP
D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D002784:Cholesterol / Q000378:metabolism; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D020675:Peroxisomes / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar
D007659:Ketones; D002784:Cholesterol; D000101:Acetyl-CoA C-Acetyltransferase