Abstract
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.
Highlights
These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity
Difference between the two pathways is that the mitochondrial pathway is initiated by a fatty acyl-CoA dehydrogenase, while the first step in the peroxisomal pathway is catalyzed by a fatty acyl-CoA oxidase (FAO) that transfers electrons directly to O2 to form H202 [1, 5, 6]
Another difference between the mitochondrial and peroxisomal @-oxidationsystems is in their chain length specificities for fatty acyl-CoAs, with the mitochondrial system being more active towards short chain substrates and the peroxisomal system utilizing exclusively medium to long chain fatty acyl-CoAs
Summary
These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. T h e results reported here for rat liver homogenates show that the absolute activity, enzyme properties, and subcellular distribution of the oxidase as assayed with the fluorometric procedure agree well with those obtained with other assay methods.
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