Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1985 Apr 1;227(1):205–210. doi: 10.1042/bj2270205

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

G M Small, K Burdett, M J Connock
PMCID: PMC1144827  PMID: 3994682

Abstract

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.

Full text

PDF
205

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bronfman M., Inestrosa N. C., Leighton F. Fatty acid oxidation by human liver peroxisomes. Biochem Biophys Res Commun. 1979 Jun 13;88(3):1030–1036. doi: 10.1016/0006-291x(79)91512-2. [DOI] [PubMed] [Google Scholar]
  2. Connock M. J., Perry S. R. Detection of acyl-CoA beta-oxidation enzymes in peroxisomes (microperoxisomes) of mouse heart. Biochem Int. 1983 Apr;6(4):545–551. [PubMed] [Google Scholar]
  3. Hryb D. J., Hogg J. F. Chain length specificities of peroxisomal and mitochondrial beta-oxidation in rat liver. Biochem Biophys Res Commun. 1979 Apr 27;87(4):1200–1206. doi: 10.1016/s0006-291x(79)80034-0. [DOI] [PubMed] [Google Scholar]
  4. Inestrosa N. C., Bronfman M., Leighton F. Detection of peroxisomal fatty acyl-coenzyme A oxidase activity. Biochem J. 1979 Sep 15;182(3):779–788. doi: 10.1042/bj1820779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kramar R., Hüttinger M., Gmeiner B., Goldenberg H. Beta-oxidation in peroxisomes of brown adipose tissue. Biochim Biophys Acta. 1978 Dec 22;531(3):353–356. doi: 10.1016/0005-2760(78)90217-5. [DOI] [PubMed] [Google Scholar]
  6. Köchli H., Von Wartburg J. P. A sensitive photometric assay for monoamine oxidase. Anal Biochem. 1978 Jan;84(1):127–135. doi: 10.1016/0003-2697(78)90491-8. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Lazarow P. B., De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2043–2046. doi: 10.1073/pnas.73.6.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lazarow P. B. Rat liver peroxisomes catalyze the beta oxidation of fatty acids. J Biol Chem. 1978 Mar 10;253(5):1522–1528. [PubMed] [Google Scholar]
  10. Le Hir M., Dubach U. C. Peroxisomal and mitochondrial beta-oxidation in the rat kidney: distribution of fatty acyl-coenzyme A oxidase and 3-hydroxyacyl-coenzyme A dehydrogenase activities along the nephron. J Histochem Cytochem. 1982 May;30(5):441–444. doi: 10.1177/30.5.7200500. [DOI] [PubMed] [Google Scholar]
  11. Murphy P. A., Krahling J. B., Gee R., Kirk J. R., Tolbert N. E. Enzyme activities of isolated hepatic peroxisomes from genetically lean and obese male mice. Arch Biochem Biophys. 1979 Mar;193(1):179–185. doi: 10.1016/0003-9861(79)90021-3. [DOI] [PubMed] [Google Scholar]
  12. Peters T. J., Shio H. Analytical subcellular fractionation studies on rat liver and on isolated jejunal enterocytes with special reference to the separation of lysosomes, peroxisomes and mitochondria. Clin Sci Mol Med. 1976 May;50(5):355–366. doi: 10.1042/cs0500355. [DOI] [PubMed] [Google Scholar]
  13. Small G. M., Brolly D., Connock M. J. Palmityl-CoA oxidase: detection in several guinea pig tissues and peroxisomal localisation in mucosa, of small intestine. Life Sci. 1980 Nov 10;27(19):1743–1751. doi: 10.1016/0024-3205(80)90441-5. [DOI] [PubMed] [Google Scholar]
  14. Small G. M., Hocking T. J., Strudee A. P., Burdett K., Connock M. J. Enhancement by dietary clofibrate of peroxisomal palmityl-CoA oxidase in kidney and small intestine of albino mice and liver of genetically lean and obese mice. Life Sci. 1981 Apr 27;28(17):1875–1882. doi: 10.1016/0024-3205(81)90293-9. [DOI] [PubMed] [Google Scholar]
  15. Walusimbi-Kisitu M., Harrison E. H. Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. J Lipid Res. 1983 Aug;24(8):1077–1084. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES