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PHoToPHoSPHoRYLATIoN AT VARIABLE ADP CoNCENTRATIoN BUT CoNSTANT DELTA-PH IN LETTUCE THYLAKoIDS - EFFECT oF DELTA-PH AND PHoSPHATE oN THE APPARENT AFFINITY FoR ADP

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BIoCHIMICA ET BIoPHYSICA ACTA 1989年第1期974卷 104-113页

作者： BIZoUARN, T DEKoUCHKoVSKY, Y HARAUX, F CNRS UPR 39 BATIMENT 24 F-91198 GIF SUR YVETTE FRANCE

There is a wide variation in the literature of the apparent affinity of membrane-bound F0F1-ATPases for the substrates of phosphorylation, especially for ADP. Since these measurements are inescapably biased by an increased drop of .DELTA.~.mu.H+ when the phosphorylating flow rises with substrate concentration, we have developed a method to keep the proton gradient constant when substrates are varied. Light-induced trapping of hexylamine inside lettuce thylakoids delays the .*** decrease when the membrane H+-conductivity increases. This was used to measure the initial rate of ATP synthesis just after the addition of variable amounts of ADP, without a significant change of the proton gradient due to the phosphorylating H+-flow. .*** was estimated by the quenching of 9-aminoacridine fluorescence, calibrated with the ''phosphate potential;>p (in State 4), and the rate of phosphorylation was computed form the medium alkalinization using a glass electrode. Saturation curves (rate vs. ADP concentration) at iso-.*** were then obtained, and the magnitude of the proton gradient was also adjusted at different levels by varying the light intensity. An inhibitory effect of high concentrations of ADP on ATP synthesis was noticed in the case of a low .***. Within all the present .*** range, phosphate, which raises Vmac, augments the apparent affinity of the coupling factor for the substrated ADP, i.e. Km is lowered. Finally, the Km for ADP obeys a complex law with the .*** increase: first, Km slightly decreases down to some kind in plateau (Km - 6-7 .mu.M around .*** 3.4), then sharply increases (Km > 20 .mu.M at .DELTA. pH .gtoreq. 3.5). In the same condition, Vmax reguarly rises, that is Vmax/Km declines for high .*** values. This cannot be explained by the classical energetic role of the protonmotive force. Rather it could reflect a regulation of substrate affinity by .***, a process also distinct from the well-known ATPase activatio

关键词： Photophosphorylation ATPase Thylakoid membrane Proton gradient Enzyme regulation: (Lettuce chloroplasts) Chl chlorophyll Δũ H +, δpH, δψ transmembrane difference of proton electrochemical potential, of pH, of electrical potential ΔG G p Gibbs’ free enthalpy of phosphorylation or ‘phosphate potential’, here at static head (‘State 4’) Δ G p o′ standard phosphate potential V , S rate, substrate of a reaction at a given ΔpH V p , V max actual or maximal rate of ATP synthesis for given or saturating ADP at set P i and ΔpH K m apparent Michaelis constant for ADP at set P i and ΔpH V e volume of the external medium V i operational (probe-sensitive) internal volume of the thylakoids CF coupling factor or ATPase

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THE BINDING AND RELEASE oF THE INHIBIToR PRoTEIN ARE GoVERNED INDEPENDENTLY BY ATP AND MEMBRANE-PoTENTIAL IN oX-HEART SUBMIToCHoNDRIAL VESICLES

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BIoCHIMICA ET BIoPHYSICA ACTA 1988年第1期933卷 12-21页

作者： LIPPE, G SoRGATo, MC HARRIS, DA UNIV PADUA INST BIOCHEM I-35100 PADUA ITALY UNIV PADUA CNR STUDY MITOCHONDRIAL PHYSIOL UNIT I-35100 PADUA ITALY UNIV OXFORD DEPT BIOCHEM OXFORD ENGLAND

(1) The effects of membrane potential (.DELTA..psi.) and nucleotides on the interaction between the F1-ATP synthase and its natural inhibitor protein (IF1) are studied in ox-heart submitochondrial vesicles. (2) Membrane potential causes displacement of IF1 from submitochondrial vesicles, as shown by measuring both .DELTA..psi.-dependent stimulation of ATPase capacity and release of 125I-labelled IF1 from the vesicles. These effects are abolished if ATP is included in the incubation. (3) There is a linear increase in the steady-state ATPase capacity of oxidising vesicles as .DELTA..psi. is increased from 100 mV to 135 mV. Increasing .DELTA..psi. above 140 mV leads to no further change. (4) At a constant membrane potential, ATP suppresses the increase in ATPase capacity, with a concentration for half maximal effect of 140 .mu.M. This value is close to the Km for ATP hydrolysis by membrane-bound F1. This suppression is related to ATP concentrations rather than to .*** or ATP/ADP ratio. (5) The unidirectional on- and off-rates of IF1 were measured separately. The off-rate of IF1 is increased by membrane potential but unaffected by ATP. The on-rate, conversely, is increased by ATP. Thus, the suppression of the potential-dependent net release of IF1 from submitochondrial vesicles by ATP results from an increase of the IF1 on-rate above the off-rate.

关键词： Inhibitor protein ATP synthase ATP synthase inhibitor protein interaction ATP Membrane potential Bovine heart submitochondrial vesicle IF 1 naturally occurring inhibitor protein of mitochondrial ATPase [ 125 I]IF 1 radioiodinated inhibitor protein MgATP SMP submitochondrial vesicles prepared in the presence of ATP and magnesium [ 125 I]IF 1 SMP submitochondrial vesicles supplemented with [ 125 I]IF 1 Δp protonmotive force = Δ∼μ H F = Δψ + (2.303 RT F ) ΔpH , where Δψ is the electrical component and ΔpH the chemical component of the transmembrane difference of the electrochemical potential of protons ( Δ ∼ μ H ) P i inorganic phosphate ΔG P = ΔG o′ + RT In ( [ATP] [ADP][P i ] ) Ap 5 A, P 1 ,P 5 -di(adenosine-5′-)pentaphosphate FCCP carbonylcyanide p -trifluoromethoxyphenylhydrazone CCCP carbonylcyanide m -chlorophenylhydrazone

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