Beard W A, Dilley R A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
J Bioenerg Biomembr. 1988 Feb;20(1):85-106. doi: 10.1007/BF00762139.
The great sensitivity of the luciferin-luciferase ATP detection system allows direct observation of ATP formation derived from single-turnover flashes in a thylakoid reaction mixture. The method can measure the energization threshold--the number of flashes required for the initiation of ATP formation--as well as detect post-illumination ATP formation after the last flash of a flash sequence. We describe the characteristics of this post-illumination phosphorylation which can be observed after a series of phosphorylating flashes (PIP+) or when the assay for ATP formation was performed in a "traditional" manner where the ADP and Pi were added after the flash-energization period (PIP-). Comparing PIP+ yields and kinetics of the PIP+ decay under various treatments can give information about membrane energization events only if it is clearly established that different PIP+ yields and decay rates are not due to limitations of the luciferase-catalyzed reaction. Experiments showing that the PIP+ ATP yield and kinetics were due to membrane-limited deenergization events (proton efflux) rather than luciferase limitations include: (1) An uncoupler, nigericin, added after the last flash reduced the PIP+ yield, but had no effect on the luciferase reaction. (2) The kinetics of the luminescence after adding standard ATP were much faster than the PIP+ kinetics. (3) Valinomycin and K+ stimulated the PIP+ yield but had no influence on the luciferase reaction. (4) Lowering the pH from 8 to 7 increased both the PIP- (an assay independent of luciferase kinetics) and the PIP+ ATP yields, an expected result owing to the greater endogenous buffering power encountered by the proton gradient when the external pH is 7. In spite of the last-mentioned point, the threshold flash number for ATP formation onset was the same for pH 7 and 8 (valinomycin, K+ present) at slow flash frequencies. This is consistent with a membrane-localized rather than a delocalized gradient. The accompanying reports (W. A. Beard, G. Chiang and R. A. Dilley, and W. A. Beard and R. A. Dilley, J. Bioenerg. Biomembr.) show that different conditions can lead to observing either localized or delocalized proton gradient coupling in the PIP+ event and the ATP onset threshold flash number.
荧光素-荧光素酶ATP检测系统的高灵敏度使得能够直接观察类囊体反应混合物中单周转闪光产生的ATP形成。该方法可以测量能量化阈值——启动ATP形成所需的闪光次数——以及检测闪光序列最后一次闪光后光照后ATP的形成。我们描述了这种光照后磷酸化的特征，这可以在一系列磷酸化闪光（PIP+）后观察到，或者当以“传统”方式进行ATP形成测定时观察到，即在闪光激发期后添加ADP和Pi（PIP-）。只有在明确确定不同的PIP+产量和衰减率不是由于荧光素酶催化反应的限制时，比较不同处理下PIP+的产量和PIP+衰减的动力学才能提供有关膜能量化事件的信息。表明PIP+ATP产量和动力学是由于膜限制的去能量化事件（质子外流）而非荧光素酶限制的实验包括：（1）在最后一次闪光后添加解偶联剂尼日利亚菌素会降低PIP+产量，但对荧光素酶反应没有影响。（2）添加标准ATP后发光的动力学比PIP+动力学快得多。（3）缬氨霉素和K+刺激PIP+产量，但对荧光素酶反应没有影响。（4）将pH从8降至7会增加PIP-（一种与荧光素酶动力学无关的测定）和PIP+ATP产量，这是一个预期结果,因为当外部pH为7时质子梯度遇到更大的内源性缓冲能力。尽管有最后一点，但对于pH 7和8（存在缬氨霉素、K+），在慢闪光频率下ATP形成开始的阈值闪光次数是相同的。这与膜定位而非离域梯度一致。随附的报告（W. A. Beard、G. Chiang和R. A. Dilley，以及W. A. Beard和R. A. Dilley，《生物能量与生物膜杂志》）表明，不同条件下可能会观察到PIP+事件中局部或离域质子梯度偶联以及ATP开始阈值闪光次数。
