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.

The composite pristanoyl‐CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5′‐RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS‐gel electrophoresis. The amino acid identity with rat palmitoyl‐CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl‐CoA oxidases (20%), suggesting that the palmitoyl‐CoA oxidase/pristanoyl‐CoA oxidase duplication occurred early in evolution. The carboxy‐terminal tripeptide of pristanoyl‐CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal‐targeting signal‐1 import receptor indicated that SQL functions as a peroxisome‐targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl‐CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl‐CoA oxidase gene, if present, is only poorly or not transcribed.

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