Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity.
Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity.
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验证用于测量膜相关 Mg-和 NaK-ATP 酶活性的记录分光光度法。
DOI：
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发表时间：
1979
期刊：
影响因子：
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通讯作者：
Ockner Rk
中科院分区：
文献类型：
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作者：
B. Scharschmidt;Keeffe Eb;N. Blankenship;Ockner Rk
测定膜相关的Mg-和na -ATP酶活性的常规方法通常包括酶与ATP的定时孵育，然后测量释放的Pi。它们耗时，需要大量的酶蛋白，并且在重复的样品中表现出相当大的可变性。这些问题可以通过偶联酶分析在很大程度上消除，其中atp酶形成ADP与NADH氧化结合，中间酶PK和LDH和中间底物磷酸烯醇丙酮酸过量存在。在340 nm处连续记录NADH氧化，NaK-ATPase代表总atp酶的抑制部分。该方法可以准确测定与酶蛋白线性变化的初始反应速率。此外，它消除了测量Pi的需要，并防止了抑制性ADP的积累。在100多种从大鼠制备的LPM制剂中，用已知的改变atp酶活性的药物预处理或暴露于体外的atp酶抑制剂。偶联酶法测定的atp酶活性与常规方法所得结果相似。此外，偶联酶测定法需要更少的膜蛋白，在重复样品中显示出更小的可变性，只需要一半的时间，并且在脑、肾和心脏组织中也能得到准确的值。这种改进的分析方法应该在膜atp酶的研究中找到广泛的应用，因为它们与各种细胞功能有关。
Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.