Estimation of peroxisomal beta-oxidation in rat heart by a direct assay of acyl-CoA oxidase.
The contribution of peroxisomes to palmitate beta-oxidation in rat heart was estimated by either inhibiting mitochondrial beta-oxidation or measuring the activity of acyl-CoA oxidase. When respiratory inhibitors such as KCN or antimycin plus rotenone, or inhibitors of mitochondrial fatty acid uptake such as 2-tetradecylglycidic acid or 2-bromopalmitate, were used, degrees of inhibitions ranging from 24% to 87% were observed for palmitate beta-oxidation by a rat heart homogenate. Although the oxidation of palmitoyl-L-carnitine by coupled rat heart mitochondria was almost completely (94%) inhibited by KCN, the inhibition by antimycin plus rotenone was incomplete (77%) and was stimulated by L-carnitine. A direct assay of acyl-CoA oxidase, based on the spectrophotometric measurement at 300 nm of 2,4-decadienoyl-CoA formation from 4-trans-decenoyl-CoA, was evaluated with the aim of obtaining reliable values for the activity of this enzyme, which is presumed to catalyse the rate-limiting step of peroxisomal beta-oxidation. Activities determined by use of this assay were much higher than activities obtained by a coupled assay [Small, Burdett and Connock (1985) Biochem. J. 227, 205-210] commonly used to measure the activity of acyl-CoA oxidase. However, both methods yielded the same relative activities with different tissue homogenates. Based on an estimated palmitoyl-CoA oxidase activity of 0.3 nmol/min per mg of protein, the contribution of peroxisomes to palmitate beta-oxidation in a rat heart homogenate would optimally be 4%, and most likely is several-fold lower.
Chu
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Department of Chemistry, City College of City University of New York, NY 10031.
Mao
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Schulz
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D042962:Acyl-CoA Oxidase; D000818:Animals; D005227:Fatty Acids / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008830:Microbodies / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D005227:Fatty Acids; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase