Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta), using spectrophotometric methods

BACKGROUND: This study was aimed primarily at testing in the liver of brown trout (Salmo trutta) spectrophotometric methods previously used to measure the activities of catalase and hydrogen peroxide producing oxidases in mammals. To evaluate the influence of temperature on the activities of those peroxisomal enzymes was the second objective. A third goal of this work was the study of enzyme distribution in crude cell fractions of brown trout liver. RESULTS: The assays revealed a linear increase in the activity of all peroxisomal enzymes as the temperature rose from 10° to 37°C. However, while the activities of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slightly affected. A crude fraction enriched with peroxisomes was obtained by differential centrifugation of liver homogenates, and the contamination by other organelles was evaluated by the activities of marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (aryl sulphatase) and microsomes (NADPH cytochrome c reductase). For peroxisomal enzymes, the activities per mg of protein (specific activity) in liver homogenates were strongly correlated with the activities per g of liver and with the total activities per liver. These correlations were not obtained with crude peroxisomal fractions. CONCLUSIONS: The spectrophotometric protocols originally used to quantify the activity of mammalian peroxisomal enzymes can be successfully applied to the study of those enzymes in brown trout. Because the activity of all studied peroxisomal enzymes rose in a linear mode with temperature, their activities can be correctly measured between 10° and 37°C. Probably due to contamination by other organelles and losses of soluble matrix enzymes during homogenisation, enzyme activities in crude peroxisomal fractions do not correlate with the activities in liver homogenates. Thus, total homogenates will be used in future seasonal and toxicological studies of brown trout peroxisomes.