Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta), using spectrophotometric methods.
Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta), using spectrophotometric methods.
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DOI：
10.1186/1471-2091-4-2
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发表时间：
2003-03-11
期刊：
影响因子：
--
通讯作者：
Lobo-da-Cunha A
中科院分区：
文献类型：
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作者：
Rocha MJ;Rocha E;Resende AD;Lobo-da-Cunha A
本研究的主要目的是测试褐鳟（Salmo trutta）肝脏中的分光光度法，该分光光度法以前用于测量哺乳动物过氧化氢酶和过氧化氢生成氧化酶的活性。第二个目的是评价温度对这些过氧化物酶体活性的影响。本工作的第三个目标是研究褐鳟肝脏粗细胞组分中的酶分布。实验结果显示，当温度从10°C升高到37°C时，所有过氧化物酶体酶的活性呈线性增加。过氧化氢氧化酶活性受温度影响较大，过氧化氢酶活性受温度影响较小。通过对肝脏均质液进行差速离心，获得了富含过氧化物酶体的粗馏分，并通过线粒体（琥珀酸脱氢酶）、溶酶体（芳基硫酸酶）和微粒体（NADPH细胞色素c还原酶）标记酶的活性来评估其他细胞器的污染。对于过氧化物酶，肝脏匀浆中每毫克蛋白质的活性（比活性）与每克肝脏的活性和每肝脏的总活性密切相关。这些相关性在粗过氧化物酶体分数中没有得到。最初用于定量哺乳动物过氧化物酶活性的分光光度法可以成功地应用于褐鳟中这些酶的研究。由于所研究的所有过氧化物酶体酶的活性都随温度线性上升，因此它们的活性可以在10°至37°C之间正确测量。可能是由于在匀浆过程中受到其他细胞器的污染和可溶性基质酶的损失，粗过氧化物酶体组分中的酶活性与肝脏匀浆中的活性不相关。因此，在未来的季节性和毒理学研究中将使用全匀浆进行褐鳟过氧化物酶体。
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. 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. 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.