菠菜叶绿体中磷酸化作用、电子流与内部氢离子浓度之间的定量关系。

Quantitative relationships between phosphorylation, electron flow, and internal hydrogen ion concentrations in spinach chloroplasts.

作者信息

Portis A R, McCarty R E

出版信息

J Biol Chem. 1976 Mar 25;251(6):1610-7.

PMID:3501

Abstract

Further evidence that the uptake of [14C]hexylamine, determined by centrifugal filtration of spinach chloroplast thylakoids through silicone fluid layers, gives precise estimations of light-induced H+ concentration gradients (deltapH) is presented. DeltapH was independent of the amount of thylakoids used or of the concentration of hexylamine. Moreover, hexylamine uptake was sensitive to the osmolarity of the suspending medium. 2. Internal H+ concentration ([H+]in) is proportional to the rate of electron flow when light intensity was used to vary these parameters. Proportionality was still observed in the presence of 0.1 and 1.0 muM gramicidin D. When, however, [H+]in and electron flow were varied by increasing the concentration of gramicidin D, at constant light intensity the rate of electron flow was approximately proportional to 1/[H]in. 3. The phosphorylation efficiency (P/e2 ratio) falls with decreasing light intensity or increasing concentrations of the phosphorylation inhibitor, 4'-deoxyphlorizin. The proportionality between the rate of electron flow and [H+]in allows the calculation of the rate of nonphosphorylating (basal) electron flow if [H+]in under phosphorylating conditions is known. The contribution of basal electron flow, a consequence of passive efflux of H+ from the thylakoids, to the overall rate of electron flow increases as the rate of phosphorylation decreases. P/e2 ratios calculated using rates of electron flow from which the basal component has been subtracted are constant. A calculated P/e2 ratio of about 1.3 is obtained. 4. It is shown that the reciprocal of the phosphorylation efficiency should be proportional to 1/[H+]in2 when these parameters are varied using light intensity. This relationship was verified and provided an estimate of the P/e2 at infinite [H+]in. This value was 1.3. These results provide further evidence that a H+ electrochemical gradient serves to couple photophosphorylation to electron flow and that the rate of phosphorylation is proportional to [H+]in3. That is, three H+ are translocated out of thylakoids for each adenosine triphosphate formed.

摘要

本文提供了进一步的证据，表明通过硅胶液层对菠菜叶绿体类囊体进行离心过滤测定的[14C]己胺摄取量，能够精确估算光诱导的H⁺浓度梯度（ΔpH）。ΔpH与所用类囊体的量或己胺的浓度无关。此外，己胺摄取对悬浮介质的渗透压敏感。2. 当用光强度改变这些参数时，内部H⁺浓度（[H⁺]in）与电子流速率成正比。在存在0.1和1.0 μM短杆菌肽D的情况下仍观察到这种比例关系。然而，当通过增加短杆菌肽D的浓度来改变[H⁺]in和电子流时，在恒定光强度下，电子流速率大约与1/[H⁺]in成正比。3. 磷酸化效率（P/e2比值）随光强度降低或磷酸化抑制剂4'-脱氧根皮苷浓度增加而下降。如果已知磷酸化条件下的[H⁺]in，电子流速率与[H⁺]in之间的比例关系允许计算非磷酸化（基础）电子流速率。基础电子流是H⁺从类囊体被动外流的结果，随着磷酸化速率降低，其对总电子流速率的贡献增加。使用减去基础成分后的电子流速率计算的P/e2比值是恒定的。得到的计算P/e2比值约为1.3。4. 结果表明，当用光强度改变这些参数时，磷酸化效率的倒数应与1/[H⁺]in²成正比。这种关系得到了验证，并提供了在无限[H⁺]in时的P/e2估计值。该值为1.3。这些结果提供了进一步的证据，表明H⁺电化学梯度用于将光合磷酸化与电子流偶联，并且磷酸化速率与[H⁺]in³成正比。也就是说，每形成一个三磷酸腺苷，有三个H⁺从类囊体转运出来。