Hydroxyeicosatetraenoic acid oxidation in Chinese hamster ovary cells: a peroxisomal metabolic pathway.
To evaluate the peroxisomal requirement for beta-oxidation of hydroxyeicosatetraenoic acids (HETES), we tested 5-, 12- and 15-HETE oxidation in wild-type and mutant Chinese hamster ovary (CHO) cells. Mutant CHO cells contain peroxisomal ghosts, have random cytosolic localization of catalase and lack two of the enzymes necessary for peroxisomal beta-oxidation. Reverse-phase HPLC indicated that 33% of 12-HETE radioactivity was converted by wild-type CHO cells during a 2 h incubation to one major and several minor polar metabolites. Wild-type CHO cells also converted 15-HETE to one major and several minor polar metabolites. Neither 12- nor 15-HETE were converted to any metabolites by the mutant CHO cell lines, despite appreciable cellular uptake of these hydroxyeicosanoids. 5-HETE was not converted to any metabolic products by either the wild-type or the mutant CHO cells. Docosahexaenoic acid beta-oxidation was substantially reduced in the mutants as compared to the wild-type cells, palmitic acid beta-oxidation was reduced to an intermediate extent in the mutants, but octanoate beta-oxidation and citrate synthase activity were not impaired. Protein immunoblotting for mitochondrial manganese superoxide dismutase indicated a single band of identity at 20 kDa in both wild-type and mutant CHO cells. Since mutant CHO cells fail to convert 12- and 15-HETE to oxidative metabolites but contain normal mitochondrial enzymatic activities, intact peroxisomes appear to be the organelle responsible for HETE oxidation.
Gordon
J A
JA
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Zoeller
R A
RA
Spector
A A
AA
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002950:Citrate (si)-Synthase / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D008830:Microbodies / Q000378:metabolism*; D009154:Mutation; D010053:Ovary / Q000166:cytology; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000378:metabolism
D005227:Fatty Acids; D006893:Hydroxyeicosatetraenoic Acids; D013482:Superoxide Dismutase; D002950:Citrate (si)-Synthase