Properties and Subcellular Localization of Myocardial Fatty Acyl-coenzyme A Oxidase

The properties and subcellular localization of fatty acyl-CoA oxidase (FAO) were studied in rat heart homogenates. After differential centrifugation, FAO was sedimentable and enriched in a “light-mitochondrial” fraction. FAO had a pH optimum of 8–9. Among straight-chain, saturated fatty acyl-CoAs, the enzyme showed a marked preference for medium chain substrates (C12 \u3e C10 = C8 \u3e C16 = C14 \u3e C6) over a concentration range up to 100 µM. No activity was observed with C4-CoA. The apparent Michaelis constant (Km) for C12-CoA was 5-10 µM. After removal of nuclei by low-speed centrifugation, combined subcellular particle preparations were obtained by high-speed centrifugation and layered on linear density gradients of metrizamide. After density equilibration, FAO showed a symmetric distribution centered at ρ = 1.16–1.18, like that of the enzyme catalase, a marker for microperoxisomes. In contrast, enzyme markers for mitochondria, lysosomes, sarcolemma, and sarcoplasmic reticulum were recovered in low-density regions of the gradient. These results provide a direct demonstration of fatty acyl-CoA oxidase in cardiac tissue and its association with microperoxisomes

Properties and Subcellular Localization of Myocardial Fatty Acyl-coenzyme A Oxidase

The properties and subcellular localization of fatty acyl-CoA oxidase (FAO) were studied in rat heart homogenates. After differential centrifugation, FAO was sedimentable and enriched in a “light-mitochondrial” fraction. FAO had a pH optimum of 8–9. Among straight-chain, saturated fatty acyl-CoAs, the enzyme showed a marked preference for medium chain substrates (C12 \u3e C10 = C8 \u3e C16 = C14 \u3e C6) over a concentration range up to 100 µM. No activity was observed with C4-CoA. The apparent Michaelis constant (Km) for C12-CoA was 5-10 µM. After removal of nuclei by low-speed centrifugation, combined subcellular particle preparations were obtained by high-speed centrifugation and layered on linear density gradients of metrizamide. After density equilibration, FAO showed a symmetric distribution centered at ρ = 1.16–1.18, like that of the enzyme catalase, a marker for microperoxisomes. In contrast, enzyme markers for mitochondria, lysosomes, sarcolemma, and sarcoplasmic reticulum were recovered in low-density regions of the gradient. These results provide a direct demonstration of fatty acyl-CoA oxidase in cardiac tissue and its association with microperoxisomes

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 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

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 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