title: Purification and further characterization of peroxisomal trihydroxycoprostanoyl-CoA oxidase from rat liver.
abstract: The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.
authors.affiliation: Katholieke Universiteit Leuven, Afdeling Farmakologie, Belgium.
authors.forename: P P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.forename: P
authors.initials: P
authors.lastname: Van Rompuy
authors.forename: J C
authors.initials: JC
authors.lastname: Vanhooren
authors.forename: G P
authors.initials: GP
authors.lastname: Mannaerts
mesh_terms: D042962:Acyl-CoA Oxidase; D000818:Animals; D008099:Liver / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
chemical_list: D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; C063427:trihydroxycoprostanoyl-CoA oxidase