DETERMINATION OF H+/ATP STOICHIOMETRY FOR THE PLASMA-MEMBRANE H+-ATPASE FROM RED BEET (BETA-VULGARIS L) STORAGE TISSUE
DETERMINATION OF H+/ATP STOICHIOMETRY FOR THE PLASMA-MEMBRANE H+-ATPASE FROM RED BEET (BETA-VULGARIS L) STORAGE TISSUE
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DOI：
10.1104/pp.95.1.242
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发表时间：
1991-01-01
期刊：
影响因子：
7.4
通讯作者：
REYNOLDSNIESMAN, I
中科院分区：
文献类型：
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作者：
BRISKIN, DP;REYNOLDSNIESMAN, I
测定了红甜菜(Beta uggaris L.，var Detroit Dark Red)贮藏组织中与天然小泡相关的质膜H+-ATPase的H+/ATP化学计量比。H+/ATP化学计量比的测定采用动力学方法，将三种不同方法估算的H+内流速率与相同条件下偶联酶试验测得的ATP水解率进行比较。这些估计H+内流的方法要么是基于从pH 6.13确定外部介质的初始碱化速率，要么是基于停止H+-ATPase后从稳定的pH梯度测量H+泄漏速率，或者基于描述H+在任何给定时间点的净运输的数学模型。当用这些方法估计的H+内流速率与ATP水解速率相比较时，观察到H+/ATP化学计量比约为1。考虑到ATP水解的最大自由能(Delta-G(ATP))，这个H+/ATP化学计量比的值足以解释在体内观察到的跨质膜的质子电化学梯度的大小。
The H+/ATP stoichiometry was determined for the plasma membrane H+-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H+/ATP stoichiometry utilized a kinetic approach where rates of H+ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H+ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H+ leakage from a steady-state pH gradient after stopping the H+-ATPase or utilizing a mathematical model which describes the net transport of H+ at any given point in time. When the rate of H+ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H+/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (DELTA-G(ATP)), this value for H+/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.