Large-scale purification and further characterization of rat pristanoyl-CoA oxidase.
The elution of pristanoyl-CoA oxidase from butyl-Sepharose required unusually high concentrations of ethylene glycol, enabling the large-scale purification of this oxidase in a single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P.P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G. P. (1991) J. Biol. Chem. 266, 24676-24683), migrated as a 513-kDa protein during native gel electrophoresis. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/mol enzyme. Its amino acid composition was different from those of other known acyl-CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl-CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2-methylpalmitoyl-CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clear signal only in rat liver, possibly indicating that pristanoyl-CoA oxidase might be a rat-specific oxidase.
Katolieke Universiteit Leuven, Campus Gasthuisberg, Afdeling Farmakologie, Belgium.
P P
PP
Van Veldhoven
P
P
Van Rompuy
M
M
Fransen
B
B
De Béthune
G P
GP
Mannaerts
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C066272:pristanoyl-CoA oxidase