Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. II. Stimulation by a K+ diffusion potential.

Addition of valinomycin, nonactin, or monactin plus KCl in the dark to preilluminated chromatophores induced the synthesis of a large amount of ATP. This stimulation of postillumination ATP synthesis by a dark-imposed K+ diffusion potential was different from the stimulation caused by addition of permeant anions or cations in the light, since it increases when the pH of the light stage decreased from 8.0 to 6.0. It was thus most pronounced when the chromatophores were preloaded with protons but the light-induced proton concentration gradient (deltapH) was low. Imposition of a Kplus diffusion potential resulted however in stimulation of ATP synthesis even when the light-induced deltapH was already above the threshold value required to initiate postillumination ATP synthesis. This situation was realized when valinomycin plus KCl were added in the dark to chromatophores preilluminated above pH 6.7 with thiocyanate as the permeant anion, and the amount of ATP formed was the sum of the yields obtained with each of these affectors by itself. On the other hand addition of thiocyanate together with valinomycin plus KCl in the dark led to inhibition of ATP synthesis. In this case the permeant anion could not affect the light-induced deltapH but it did eliminate the diffusion potential by decreasing the difference between the permeabilities of Kplus and the anion present in the reaction mixture.     

Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.     

delta pH driven energy-linked NAD+ reduction in Rhodospirillum rubrum chromatophores

An artificial proton gradient provided sufficient energy to drive reverse electron transport from succinate to NADH:ubiquinone oxidoreductase in chromatophores isolated from Rhodospirillum rubrum. The pH gradient created was able to reduce NAD+. In chromatophores, the optimal rate of NAD+ reduction was about 0.4-0.45 mumol NADH formed/min.mumol bacteriochlorophyll at delta pH 3. The presence of oligomycin was an obligate factor in the assay in order to observe the maximal rate of NAD+ reduction. The rate of NADH formation was dependent on the size of the induced pH gradient. The total NADH formed had a threshold value for the imposed delta pH. The effect of different inhibitors and uncouplers was demonstrated. Comparison between ATP, PPi, and light with the pH jump driven NAD+ reduction rate was studied.     

Light-induced proton permeability changes in retinal rod photoreceptor disk membranes.

We have used the membrane-permeant charged fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide (diS-C3[5]), to monitor electrical potentials across the membranes of isolated bovine disks. Calibration curves obtained from experiments where a potential was created across the disk membrane by a potassium concentration gradient and valinomycin showed an approximately linear relation between dye fluorescence and calculated membrane potential from 0 to -120 mV. Light exposure in the presence of the permeant buffer, imidazole, caused a rapid decay of the membrane potential to a new stable level. Addition of CCCP, a proton ionophore, in the dark produced the same effect as illumination. When the permeant buffer, imidazole, was replaced by the impermeant buffer, Hepes, neither light nor CCCP discharged the gradient. We interpret the changes in membrane potential measured upon illumination to be the result of a light-induced increase in the permeability of the disk membrane to protons. A permeant buffer is required to prevent the build-up of a pH gradient which would inhibit the sustained proton flow needed for an observable change in membrane potential.     

Electrochemical gradients and energy coupling in photosynthetic bacteria

The electrochemical proton gradient formed during light-induced electron transport in bacterial chromatophores is composed of both a proton concentration gradient and a membrane potential that can interchange under appropriate conditions. Both components, whether light-induced or imposed artificially in the dark, can drive ATP synthesis.     

Light-induced pH changes and changes in absorbance of pH indicators in Rhodospirillum rubrum chromatophores.

1. The light-induced pH change of chromatophore suspensions from Rhodospirillum rubrum was stimulated significantly and similarly by KCl, NaCl, LiCl, RbCl, CsCl, MgCl2, MnCl2, and CaCl2. In the dark, the pH of chromatophore suspensions decreased immediately and markedly on adding these salts. 2. The light-induced pH change stimulated by KCl plus valinomycin was inhibited by LiCl and NaCl, but not by RbCl. 3. The optimum pH values for light-induced pH change and photosynthetic ATP formation were around 5 and 8, respectively. The amount of chromatophore-bound ubiquinone-10 reduced in the light was independent of pH from 5 to 9. At pH 8, the number of protons incorporated into chromatophores in the light was one-half of the number of ubiquinone-10 molecules reduced in the light. 4. Among several pH indicators tested, bromothymol blue (BTB) and neutral red (NR) showed absorbance changes on illumination of chromatophores. Although the pH change indicated by the absorbance change was opposite to the light-induced pH change of the medium, the effect of KCl on the absorbance changes of BTB and NR, and the effect of valinomycin on that of NR, but not on that of BTB, were similar to those on the light-induced pH change. 5. The light-induced absorbance change of BTB was significantly inhibited by NR, whereas that of NR was hardly influenced by BTB. 6. Oligomycin stimulated the light-induced absorbance change of BTB under either non-phosphorylating or phosphorylating conditions. On the other hand, that of NR under phosphorylating conditions was 50% of that under non-phosphorylating conditions, and was increased by oligomycin.     

Effects of permeant buffers on the initiation of photosynchronous phosphorylation and postillumination phosphorylation in chloroplasts

Under canonical chemiosmotic formulations, the development of a delocalized transmembrane proton gradient should precede and, in the absence of a membrane potential, should account for all the capacity of an energy transducing system to synthesize ATP. Furthermore, any agents, such as permeant proton-absorbing buffers, that slow down the kinetics of the development of this gradient should, consequently, delay ATP synthesis. We have studied the very early (0 through 1000 ms) steps of photosynthetic ATP synthesis utilizing real-time, rapid flow-quench techniques. We have investigated the effect(s) that permeant buffers exert on this process where these buffers show no uncoupling effects, and the transmembrane potential has been collapsed by valinomycin and K+. Experimentally this system was dissected into two ATP synthesizing components, as follows: synthesis of ATP strictly concomitant with light influx and unaffected by the addition of permeant buffers. We refer to this as photosynchronous phosphorylation and synthesis of ATP monitored after the light was extinguished and which was greatly diminished by the addition of proton-absorbing permeant buffers, thus exhibiting the characteristics of conventional postillumination phosphorylation, and we suggest that it represents part of capacitance phosphorylation. The potential for capacitance phosphorylation initiates very rapidly under light and gradually builds up to steady-state level, and it is governed by canonical chemiosmotic principles. We estimate that its contribution to overall ATP yield is minimal during the first few cycles of the system and that it increases gradually towards steady state when it contributes to the majority of ATP synthesized. Neither a delocalized transmembrane proton gradient nor a strictly localized intramembrane proton pathway can account for these observations so we have proposed that a gating mechanism exists which delivers intramembrane protons initially directly to the ATP synthetase complex but subsequently to the lumen as well, and thus, allows the lumen to act as a capacitor during the steady state. This study can reconcile the findings of Ort et al. (Ort, D. R., Dilley, R. A., and Good, N. E. (1976) Biochim. Biophys. Acta 449, 108-124) with the contrasting findings of Vinkler et al. (Vinkler, C., Avron, M., and Boyer, P. D. (1980) J. Biol. Chem. 255, 2263-2266) through the opposite effects which osmotic strength and KCl concentration exert on the two ATP synthetic phases (during and after illumination) of the rapid flash technique used in those studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+/ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation.

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and Pi of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H+ consumption during the synthesis of ATP. The ratio of H+ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 H+/ATP. 2. The stimulation of the electrical potential decay by ADP and Pi was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No 'critical' or 'threshold' potential was observed. 3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle. 4. The stimulation of the electrical potential decay by ADP and Pi is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H+-release on the inside of the chromatophore vesicles following a flash.     

Comparison of the electrochemical proton gradient and phosphate potential maintained by Rhodospirillum rubrum chromatophores in the steady state.

A technique for the estimation of light-induced membrane potential in chromatophores is described. It is based on measurement of light-induced enhancement in fluorescence of 8-anilinonaphthalene sulfonic acid, which is calibrated by known K⁺ diffusion potentials. The electrochemical proton gradient (Δ_μH+^?) formed during lightinduced electron transport in Rhodospirillum rubrum chromatophores amounts to 250 mV, which is almost equally distributed between the membrane potential and the pH gradient as measured by changes in the fluorescence of anilinonaphthalene sulfonate and 9-amino acridine. Addition of the permeant anion, NaSCN, or of NH₄Cl reduces the overall Δ_μH+^? by less than 20% but changes its distribution between the pH gradient and the membrane potential so that with NaSCN it is composed mainly of the first and with NH₄Cl mainly of the second. Initiation of phosphorylation causes a drop of about 50 mV in the measured Δ_μH+^?. In the absence of salts, the drop is observed in both components, although two-thirds of it are reflected in the membrane potential. In the presence of NaSCN or NH₄Cl the 50-mV drop is exclusively recorded in the pH gradient or in the membrane potential, respectively. The steady-state phosphate potential maintained during electron transport was found to change in parallel to the Δ_μH+^?, but exceeded it by 60 to 80 mV when based on a stoichiometry of two protons translocated per ATP synthesized.     

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation.

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and P_i of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of the pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H⁺ consumption during the synthesis of ATP. The ratio of H⁺ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 $\text{H}{}^{\mathrm{+}}\text{ATP}$.2. The stimulation of the electrical potential decay by ADP and P_i was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No ‘critical’ or ‘threshold’ potential was observed.3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle.4. The stimulation of the electrical potential decay by ADP and P_i is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H⁺-release on the inside of the chromatophore vesicles following a flash.     

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

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 P_i 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.     

Isolation and purification of an active gamma-subunit of the F0.F1-ATP synthase from chromatophore membranes of Rhodospirillum rubrum. The role of gamma in ATP synthesis and hydrolysis as compared to proton translocation

We have earlier shown that extraction of Rhodospirillum rubrum chromatophores with LiCl removed completely the beta-subunit of their coupling factor ATPase complex leaving the other four subunits attached to the membrane (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8747-8752). Further treatment of these beta-less chromatophores with LiBr, under the described optimal conditions, resulted in specific removal of one additional subunit, the gamma-subunit, and both subunits were purified to homogeneity. The beta, gamma-less chromatophores as well as the beta-less ones lost their ATP-linked activities, but retained their light-induced proton uptake, resulting in the formation of an electrochemical gradient of protons composed of both a pH gradient and a membrane potential. These results indicate that the removed beta and gamma subunits cannot be an integral part of an H+ gate in the R. rubrum chromatophore membrane. Each of the removed subunits could bind to the beta, gamma-less chromatophores, but such separate reconstitution of either beta or gamma alone did not lead to restoration of any ATP-linked activity. ATP synthesis and hydrolysis could be restored to the same extent to these chromatophores by their reconstitution with both beta and gamma. It is thus concluded that the presence of both subunits is required for ATP synthesis as well as hydrolysis by the R. rubrum F0.F1 complex. The identical degree of elimination and restoration of ATP synthesis and hydrolysis upon removal and reconstitution of beta and gamma indicates that in R. rubrum at least, there seems to be no reason for suggesting the operation of different catalytic sites for the two activities.     

Quantification of the electrochemical proton gradient and activation of ATP synthase in leaves

We have developed a new method to quantify the transmembrane electrochemical proton gradient present in chloroplasts of dark-adapted leaves. When a leaf is illuminated by a short pulse of intense light, we observed that the light-induced membrane potential changes, measured by the difference of absorption (520 nm-546 nm), reach a maximum value (approximately 190 mV) determined by ion leaks that occur above a threshold level of the electrochemical proton gradient. After the light-pulse, the decay of the membrane potential follows a multiphasic kinetics. A marked slowdown of the rate of membrane potential decay occurs approximately 100 ms after the light-pulse, which has been previously interpreted as reflecting the switch from an activated to an inactivated state of the ATP synthase (Junge, W., Rumberg, B. and Schr?der, H., Eur. J. Biochem. 14 (1970) 575-581). This transition occurs at approximately 110 mV, thereby providing a second reference level. On this basis, we have estimated the Delta micro (H(+)) level that pre-exists in the dark. Depending upon the physiological state of the leaf, this level varies from 40 to 70 mV. In the dark, the Delta micro (H(+)) collapses upon addition of inhibitors of the respiratory chain, thus showing that it results from the hydrolysis of ATP of mitochondrial origin. Illumination of the leaf for a period longer than several seconds induces a long-lived Delta micro (H(+)) increase (up to approximately 150 mV) that reflects the light-induced increase in ATP concentration. Following the illumination, Delta micro (H(+)) relaxes to its dark-adapted value according a multiphasic kinetics that is completed in more than 1 h. In mature leaf, the deactivation of the Benson-Calvin cycle follows similar kinetics as Delta micro (H(+)) decay, showing that its state of activation is mainly controlled by ATP concentration.     

Photophosphorylation and oxidative phosphorylation in intact cells and chromatophores of an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh114.

Light-induced ATP synthesis was studied in intact cells and chromatophores of Erythrobacter sp. strain OCh114. ATP synthesis was measured by both the pH method and the luciferin-luciferase luminescence method. The rate of ATP synthesis was moderate (a typical value of 0.65 mol of ATP per mol of bacteriochlorophyll per min), and synthesis was inhibited by antimycin A. ATP was synthesized under illumination only under aerobic conditions and not under anaerobic conditions. This characteristic was similar to that of other light-induced energy transduction processes in this bacterial species, such as oxidation of reaction center, oxidation of cytochrome c551, and translocation of H+, which were not observed under anaerobic conditions. This phenomenon was reconciled with the fact that the Erythrobacter sp. could not grow anaerobically even in the light. The characteristics of oxidative phosphorylation and ATP hydrolysis were also investigated. The respiratory ratio of chromatophores was 2.3. Typical rates of oxidative phosphorylation by NADH and by succinate were 2.9 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.22) and 1.1 mol of ATP per mol of bacteriochlorophyll per min (P/O = 0.19), respectively. A typical rate of ATP hydrolysis was 0.25 mol of ATP per mol of bacteriochlorophyll per min in chromatophores. ATPase and adenylate kinase are also involved in the metabolism of adenine nucleotides in this bacterium.     

Sequential removal and reconstitution of subunits beta and gamma from a membrane-bound Fo.Fl-ATP synthase

The β and γ subunits of the Fo·F_l-ATP synthase complex of $\text{Rhodospirillum}\text{,}\text{rubrum}$ chromatophores were removed in two consecutive steps. The resulting depleted chromatophores lost all their ATP synthesizing activity but retained 70% of the light-induced proton uptake. ATP synthesis could be restored by reattachment of the isolated β and γ subunits together, but not of either one of them separately. These data suggest that the γ and β subunits are required for the operation of the chromatophore ATP synthase, but do not seem to participate in the light-induced proton uptake.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores.

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation. (2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl2 + phosphate, or ADP + MgCl2 + arsenate during preillumination provides full protection against this effect. (3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished. (4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate. (5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Light energy conservation processes in Halobacterium halobium cells.

In Halobacterium halobium, proton pumping driven by light or by respiration generates an electrochemical potential difference across the membrane. Energy storage in this form is only transient. Cellular energy transducers competing with proton leaks stabilize this free energy as high energy phosphate bonds, electrochemical potential of other ions, and chemical potential of amino acids and possibly other chemical species. The pH changes induced by light or by respiration in cell suspensions are complicated by proton flows associated with the functioning of the cellular energy transducers. Dominant is the proton inflow coupled to the synthesis of ATP, which has been kinetically resolved. A proton-per-ATP ratio of about 3 is calculated from simultaneous measurements of photophosphorylation and the proton inflow. This value is compatible with the chemiosmotic coupling hypothesis. The time course of the light-induced changes in membrane potential indicates that light-driven pumping increases a dark preexisting potential of about 130 mV only by a small amount (20-30 mV). The complex kinetic features of the membrane potential changes do not closely follow those of the pH changes, indicating that flows of ions other than protons are involved. A qualitative model consistent with the available data is presented. A salient feature of this model is a sudden relaxation of the protonmotive force by a proton inflow through the ATPase when the preexisting protonmotive force is increased by light or respiration and reaches a critical value. The trigger could be either the proton-motive force, the pH gradient, or possibly the internal pH.     

Superoxide dismutase catalyzes activation of synaptosomal soluble guanylate cyclase from rat brain

The hydrogen ion changes resulting from the photolysis of the rod visual pigment, rhodopsin, were investigated at acidic pH (5.2–6.5). After light-induced proton uptake, slow proton release occurred both in the dark and in the light. It was found that the amount of proton release in the dark was not equal to that in the light; about 0.9 proton remained bound to rhodopsin bleached in the dark, while all the bound protons were released in the light. Furthermore, the time course of proton release in the dark is not related to the decay of metarhodopsin II₃₈₀, but is closely related to the formation of metarhodopsin III₄₆₅.