ALPHA-OXIDATION OF 3-METHYL-SUBSTITUTED FATTY-ACIDS IN RAT-LIVER - PRODUCTION OF FORMIC-ACID INSTEAD OF CO2, COFACTOR REQUIREMENTS, SUBCELLULAR-LOCALIZATION AND FORMATION OF A 2-HYDROXY-3-METHYLACYL-COA INTERMEDIATE

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was s tudied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of alpha-oxidation is formic acid, which is then converted to CO2. Rates of alpha-oxidation identical to those observed in intac t hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and asco rbate wore added, suggesting that alpha-oxidation involves a fatty aci d activation reaction and a dioxygenase reaction. Subcellular fraction ation by differential and density gradient centrifugation demonstrated that alpha-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. alpha-Oxidation in broken cell systems went hand in hand with the formation of a 2-hy droxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confi ned to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of alpha-oxidation in intact cells, indicating that the 2- hydroxyacyl-CoA ester is a bona fide intermediate of alpha-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty a cyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. O ur experiments demonstrate that alpha-oxidation is a peroxisomal proce ss that consists of at least three reactions: fatty acid activation, h ydroxylation and the reaction(s) involved in the release of formic aci d.