A sensitive assay of acyl-coenzyme A oxidase by coupling with beta-oxidation multienzyme complex.
A highly sensitive and reliable method for assaying acyl-CoA oxidase (EC 1.3.99.3) activity was developed. An acyl-CoA oxidase-dependent [1-14C]palmitoyl-CoA degradation to acetyl-CoA, acid-soluble products, was measured by coupling with the multienzyme complex for fatty acid oxidation from Pseudomonas fragi. The activity, more than 2 pmol/min, could be assessed using this method. The activity was dependent on the coupling enzyme (multienzyme complex), coenzymes such as NAD+ and CoA, and oxygen, and the interference of acyl-CoA dehydrogenases was excluded. The activity in human samples of cultured skin fibroblasts and lymphocytes was compatible with the expected activity calculated from the amount of acyl-CoA oxidase protein estimated by immunoblot analysis. The method which was verified in several experiments can be used for clinical diagnosis of acyl-CoA oxidase deficiency and for determination of activity in samples with a low level of acyl-CoA oxidase.
Souri
M
M
Department of Biochemistry, Shinshu University School of Medicine, Nagano, Japan.
Aoyama
T
T
Hashimoto
T
T
D042962:Acyl-CoA Oxidase; D000818:Animals; D002250:Carbon Radioisotopes; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007202:Indicators and Reagents; D007700:Kinetics; D008052:Lipid Metabolism, Inborn Errors / Q000175:diagnosis; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms; D008214:Lymphocytes / Q000201:enzymology; D009097:Multienzyme Complexes; D010088:Oxidoreductases / Q000032:analysis* / Q000172:deficiency / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology; D011865:Radioisotope Dilution Technique; D051381:Rats; D012680:Sensitivity and Specificity; D012867:Skin / Q000201:enzymology; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase