Photophosphorylation as a function of illumination time. II. Effects of permeant buffers

(1) The amounts of orthophosphate, bicarbonate and tris(hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid.(2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KCl are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl)aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KCl, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrpancies occur with all of the other buffers mentioned.(3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KCl, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase.(4) We suggest that the protons produced by electron transport may be used directly for phosophorylation without ever entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.     

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms.(2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists.(3) Various ions which permeate the thylakoid membrane (K⁺ or Rb⁺ in the presence of valinomycin, SCN^?, I^?, or ClO₄^?) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K⁺ with valinomycin and the effect of SCN^? are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination.(4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.     

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)     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

The effect of permeant buffers on initial ATP synthesis by chloroplasts using rapid mix-quench techniques

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KCl are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KCl pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post-illumination ATP synthesis are driven by distinct components of the proton motive potential.     

Saccadic target selection as a function of time

Recent evidence indicates that stimulus-driven and goal-directed control of visual selection operate independently and in different time windows (van Zoest et al., 2004). The present study further investigates how eye movements are affected by stimulus-driven and goal-directed control. Observers were presented with search displays consisting of one target, multiple non-targets and one distractor element. The task of observers was to make a fast eye movement to a target immediately following the offset of a central fixation point, an event that either co-occurred with or soon followed the presentation of the search display. Distractor saliency and target-distractor similarity were independently manipulated. The results demonstrated that the effect of distractor saliency was transient and only present for the fastest eye movements, whereas the effect of target-distractor similarity was sustained and present in all but the fastest eye movements. The results support an independent timing account of visual selection.     

Nitrification in soil as a function of time

The dependence of nitrification on time may be expressed as ΣN=C/(m+1) ·t ^m+1 since its logarithmic form log ΣN=K logt+q suggests the possibility of a linear relationship between log ΣN and logt such as was found in more than 50 cases of nitrification in different soils. It was further shown that the equations for the integration curve and for the rate curve are of the same form, differing only in the constants.     

弱有机碱对光合磷酸化的影响

类囊体内部介质可被弱有机碱如吡啶、苯胺、咪唑缓冲,致使[H_(in)~ ] 减少,ΛpH幅度降低,光合磷酸化(PSP)反应被抑制,对苯二胺(p—PD)降低ΛpH,但显著促进PSP反应、在MV或PMS系统中均有此现象;在 MV DBMIB;MV DCMU或PMS DBMIB系统中,p—PDH_2重建ΛpH并恢复PSP反应活力。从p—PD(H_2)既具有弱有机碱性质,又可作为电子供体、氢递体,代替QH_2或PQH_2调节电子传递起转移质子的两种作用,解释了p—PD对ΛpH的抑制,却又促进PSP反应的现象。讨论了PSP反应对[H_(in)~ ],ΛpH幅度大小的依赖关系。     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

Stimulation of Photophosphorylation by Ascorbate as a Function of Light Intensity

When isolated, stroma-free thylakoids are illuminated in the presence of ADP and orthophosphate in the absence of any electron acceptor except O2, the addition of ascorbate stimulates electron transport through the formation of the radical monodehydroascorbate and the coupled synthesis of ATP (G. Forti and G. Elli [1995] Plant Physiol 109: 1207-1211). The stimulation is shown here to be higher at low light intensity. These observations are explained in terms of the increase of the electron transport rate by ascorbate, which established a higher value of the steady-state pH gradient, causing activation of ATP synthase, which is known to be dependent on the level of the H+-electrochemical potential difference, and a higher rate of proton flux across the membranes available for utilization by ATP synthesis.     

Vertical distribution of Atlantic salmon (Salmo salar) as a function of illumination

The vertical distribution of cultured Atlantic salmon larger than 1 kg was monitored by hydroacoustics in both a shallow (6 m) and a deep (20 m) cage. Surface light levels and fish distribution were registered for three periods throughout the year. The two fish groups were fed calculated rations twice a day. During the winter and spring (including vernal equinox), the fish were distributed at around 5 m depth in both cages when not feeding. Around summer solstice, the fish preferred deeper waters when not feeding. The light dependency of the vertical migration was pronounced, and indicates clearly that even a 20 m deep cage is not deep enough to meet the depth requirements of large Atlantic salmon during summer.     

The fluorescence of coumarin derivatives as a function of pH. II

The relative intensity and color of the fluorescence of 54 additional coumarin derivatives and of 5 new samples of compounds previously studied are reported for hydrogen-ion concentrations ranging from pH − 1.6 to 12.6. None of the pH-fluorescence curves of these derivatives are similar to those obtained with the unknown substances isolated from oat roots.     

pH gradients generated by polyprotic buffers. II. Experimental validation

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.     

Effects of dimethoate on snail B-esterase and growth as a function of dose, time and exposure route in a laboratory bioassay.

The aim was to study the effects of dimethoate on enzymatic targets and on the growth of Helix aspersa for different times and modes of exposure under laboratory conditions. Young snails were exposed to increasing dimethoate concentrations in the food (D.exp) or in an artificial substrate (S.exp) for 1, 2, 7 and 14 days. Both acetylcholinesterase (AChE) and carboxylesterase (CaE) activities were measured in the foot of the snails for each concentration and exposure time tested. Growth was evaluated after 7 days of exposure. AChE inhibition, dose-dependent for all lengths of exposure, was stronger in S.exp. AChE was more sensitive than CaE for both modes of exposure. IC50(-7) days was 38.3 micrograms g-1 in D.exp and 11.7 micrograms g-1 in S.exp for AChE and was higher than 150 micrograms g-1 in two exposure modes for CaE. AChE activity decreased from the first day to reach maximum inhibition after 7 days of exposure. As noted for B-esterase activities, growth inhibition was stronger in S.exp and was only significant for AChE inhibition of > 90%. The present results show that AChE activity could be used to give early warning of toxic effects of dimethoate in terrestrial gastropods.     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.