Measurement of ATPases in red cells: setting up and validation of a highly reproducible method.

Enzyme & protein Pub Date : 1994-01-01 DOI:10.1159/000474976
E Matteucci, F Cocci, L Pellegrini, G Gregori, O Giampietro
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引用次数: 16

Abstract

Our aim was to set up and validate a reproducible method to study ATPase family on erythrocyte membranes. We compared several methods for erythrocyte washing and hemolysis and succeeded in preparing completely hemoglobin-free membrane ghosts still bearing intact ATPases. We compared the conventional incubation procedure with the coupled enzyme assay to measure Na-K-Mg, Ca-Mg and Mg ATPase on the membranes. A significant difference was constantly observed between the results by these methods, the values by the incubation procedure being 28, 57 and 58% of the respective values obtained by the linked enzyme assay. By adopting this last one, we obtained uniform and reproducible results in 31 healthy subjects. The following activities of the measured pumps resulted: Na-K-Mg ATPase 0.026 +/- 0.007, mean +/- SD; Ca-Mg ATPase 0.030 +/- 0.010, and Mg ATPase 0.017 +/- 0.003 U/mg protein, respectively. Finally, we investigated the effect of membrane storage time and temperature on ATPase results.

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红细胞中atp酶的测定:建立和验证一种高度可重复的方法。
我们的目的是建立并验证一种可重复的方法来研究红细胞膜上的atp酶家族。我们比较了几种红细胞洗涤和溶血的方法,成功地制备了完全无血红蛋白的膜,仍然含有完整的atp酶。我们比较了传统的培养方法和偶联酶法来测定膜上的Na-K-Mg、Ca-Mg和Mg atp酶。这些方法的结果之间不断观察到显著差异,孵育程序的值分别为连接酶测定获得的值的28%,57%和58%。通过采用最后一种方法,我们在31名健康受试者中获得了一致且可重复的结果。所测泵的活性如下:Na-K-Mg atp酶0.026 +/- 0.007,平均+/- SD;Ca-Mg ATPase分别为0.030 +/- 0.010和Mg ATPase 0.017 +/- 0.003 U/ Mg蛋白。最后,我们研究了膜储存时间和温度对atp酶结果的影响。
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