Ligeti E, Brandolin G, Dupont Y, Vignais P V
Biochemistry. 1985 Jul 30;24(16):4423-8. doi: 10.1021/bi00337a025.
Phosphate-phosphate exchange through the inorganic phosphate (Pi) carrier of rat liver mitochondria was investigated by a new rapid filtration technique, which does not require the use of transport inhibitors to stop the reaction and offers high time resolution (starting from 10 ms), thus allowing kinetic measurements on a fine time scale even at room temperature. At approximately 22 degrees C, isotopic equilibrium of [32P]Pi is achieved within 0.8-2.5 s--depending on the Pi concentration--and an initial linear phase, lasting for 400-500 ms, is observed. Complete inhibition of Pi exchange by an excess (33 nmol/mg) of mersalyl, a well-known organomercurial inhibitor, required 200 ms, pointing to the insufficiency of this reagent for effective inhibitor stop. On the other hand, investigation of the effect of mersalyl (allowed to react with mitochondria for at least 20 s) on the initial rate of Pi exchange supports earlier observations on the protective effect of this inhibitor; i.e., up to 3 nmol of mersalyl/mg of protein does not decrease the transport rate whereas these low concentrations protect approximately 50% of the transport capacity from irreversible inactivation by N-ethylmaleimide. In nonrespiring mitochondria, at pH 7.3, Pi exchange exhibited a Km of 1.6 mM and a Vmax of 3.0 mumol min-1 (mg of mitochondrial protein)-1. The increase of the membrane potential without any concomitant change of delta pH had no significant influence on the kinetic parameters. The maximal velocity of Pi transport is significantly higher than the maximal velocity of all the other components of oxidative phosphorylation at comparable temperatures. The possible physiological significance of this excess capacity is discussed.
通过一种新的快速过滤技术研究了大鼠肝线粒体无机磷酸（Pi）载体介导的磷酸 - 磷酸交换，该技术无需使用转运抑制剂来终止反应，且具有高时间分辨率（起始为10毫秒），因此即使在室温下也能在精细时间尺度上进行动力学测量。在约22摄氏度时，[32P]Pi的同位素平衡在0.8 - 2.5秒内达到（取决于Pi浓度），并观察到持续400 - 500毫秒的初始线性阶段。过量（33 nmol/mg）的汞撒利（一种著名的有机汞抑制剂）完全抑制Pi交换需要200毫秒，这表明该试剂用于有效抑制终止是不足的。另一方面，研究汞撒利（使其与线粒体反应至少20秒）对Pi交换初始速率的影响支持了关于该抑制剂保护作用的早期观察结果；即，高达3 nmol的汞撒利/毫克蛋白质不会降低转运速率，而这些低浓度可保护约50%的转运能力免受N - 乙基马来酰亚胺的不可逆失活。在非呼吸状态的线粒体中，在pH 7.3时，Pi交换的Km为1.6 mM，Vmax为3.0 μmol min-1（毫克线粒体蛋白）-1。膜电位增加而ΔpH无任何伴随变化对动力学参数无显著影响。在可比温度下，Pi转运的最大速度显著高于氧化磷酸化所有其他组分的最大速度。讨论了这种过量能力可能的生理意义。
