Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity.
These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopol...
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
1983-08-01
|
| Series: | Journal of Lipid Research |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520379220 |
| _version_ | 1818727125150072832 |
|---|---|
| author | M Walusimbi-Kisitu E H Harrison |
| author_facet | M Walusimbi-Kisitu E H Harrison |
| author_sort | M Walusimbi-Kisitu |
| collection | DOAJ |
| description | These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available. |
| first_indexed | 2024-12-17T22:09:07Z |
| format | Article |
| id | doaj.art-49ec42fd7ab6421f8a55d382d8e3f445 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-17T22:09:07Z |
| publishDate | 1983-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-49ec42fd7ab6421f8a55d382d8e3f4452022-12-21T21:30:46ZengElsevierJournal of Lipid Research0022-22751983-08-0124810771084Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity.M Walusimbi-KisituE H HarrisonThese studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.http://www.sciencedirect.com/science/article/pii/S0022227520379220 |
| spellingShingle | M Walusimbi-Kisitu E H Harrison Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. Journal of Lipid Research |
| title | Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. |
| title_full | Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. |
| title_fullStr | Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. |
| title_full_unstemmed | Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. |
| title_short | Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity. |
| title_sort | fluorometric assay for rat liver peroxisomal fatty acyl coenzyme a oxidase activity |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520379220 |
| work_keys_str_mv | AT mwalusimbikisitu fluorometricassayforratliverperoxisomalfattyacylcoenzymeaoxidaseactivity AT ehharrison fluorometricassayforratliverperoxisomalfattyacylcoenzymeaoxidaseactivity |