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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.
Eur J Biochem. 1996 Sep 15; 240(3):674-83.EJ

Abstract

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was studied 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 intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and ascorbate were added, suggesting that alpha-oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation 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-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined 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 acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. Our experiments demonstrate that alpha-oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.

Authors+Show Affiliations

Katholieke Universiteit Leuven, Afdeling Farmacologie, Belgium.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8856070

Citation

Croes, K, et al. "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." European Journal of Biochemistry, vol. 240, no. 3, 1996, pp. 674-83.
Croes K, Casteels M, De Hoffmann E, et al. 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. Eur J Biochem. 1996;240(3):674-83.
Croes, K., Casteels, M., De Hoffmann, E., Mannaerts, G. P., & Van Veldhoven, P. P. (1996). 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. European Journal of Biochemistry, 240(3), 674-83.
Croes K, et al. 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. Eur J Biochem. 1996 Sep 15;240(3):674-83. PubMed PMID: 8856070.
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TY - JOUR T1 - 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. AU - Croes,K, AU - Casteels,M, AU - De Hoffmann,E, AU - Mannaerts,G P, AU - Van Veldhoven,P P, PY - 1996/9/15/pubmed PY - 1996/9/15/medline PY - 1996/9/15/entrez SP - 674 EP - 83 JF - European journal of biochemistry JO - Eur J Biochem VL - 240 IS - 3 N2 - alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was studied 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 intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and ascorbate were added, suggesting that alpha-oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation 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-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined 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 acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. Our experiments demonstrate that alpha-oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/8856070/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_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1996&volume=240&issue=3&spage=674 DB - PRIME DP - Unbound Medicine ER -