ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. I. An assay using luciferin-luciferase luminescence

Abstract

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.

Intact Chloroplasts Show Ca2+-Gated Switching between Localized and Delocalized Proton Gradient Energy Coupling (ATP Formation)

“…Samples ofsome intact plastid preparations used for the phosphorylation experiments were frozen and thawed, then diluted into 0.1% LaCl3 to further osmotically burst the organelles and displace K+ by Laj3+. The membranes were then pelleted, the supernatant boiled for 5 min, precipitated protein pelleted again, and the K+ content of the supernatant was determined by standard Atomic Absorption techniques. The total K+ measured was related back to the original plastid source assuming an aqueous plastid internal volume of 20 AL H20 (mg Chl)-'.…”

“…Single-turnover flash-induced ATP formation was measured as described by Beard 5 AM diadenosine pentaphosphate (Sigma), and 0.1 mm methyl viologen in the presence or absence of 5 mm pyridine. ATP formation was detected by measuring luminescence using the luciferin-luciferase system prepared from the Pharmacia-LKB ATP assay kit.…”

“…A 3 min incubation was given in all cases prior to starting the light flash sequence. The ATP formation onset lag was estimated as in earlier work (3,5) by the number of flashes required to elicit the first detectable rise in luminescence (45 flashes, Fig. 2E) and by extrapolation of the steady state flash yield line to the baseline (54 flashes, Fig.…”

“…Single-turnover flash-induced ATP formation can be directly and easily detected by the luciferin-luciferase assay system (3,5). With ADP and Pi present during the energization period, both the length of the ATP formation onset lag and the pIp2 ATP yield can be measured (5,6).…”

“…With ADP and Pi present during the energization period, both the length of the ATP formation onset lag and the pIp2 ATP yield can be measured (5,6). The reader should note that for better accuracy we use two parameters to measure the ATP onset lag (cf.…”