Freezing of isolated thylakoid membranes in complex media. VI. The effect of pH

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were used as a model biomembrane system for evaluating the significance of the hydrogen ion activity for cryoprotection. After freeze-thaw treatment in a buffered complex medium adjusted to various pH, light-induced photosynthetic membrane reactions were determined at optimum proton concentration. When thylakoids were suspended at hydrogen ion activities above and below the physiologically important pH range, irreversible inhibition of membrane functions was significantly less distinct after freezing at -15 degrees C than after storage for the same time at 0 degree C. It is suggested that thylakoid preservation at subfreezing temperatures could be due to temperature- and concentration-induced changes of the proton activity in the unfrozen part of the system and retardation of the temperature-dependent aging processes of the isolated membranes. In addition, the increase in the concentration of cryoprotective compounds during freezing could stabilize chloroplast membranes against the deleterious effect of unfavorable high and low proton concentrations. Thylakoid injury brought about by lowering the pH was primarily due to dissociation of the chloroplast coupling factor (CF1), which increased the proton permeability of the membranes and caused inhibition of photophosphorylation. In media adjusted to more alkaline pH, inactivation of the water oxidation system was an initial result of membrane damage. Then, noncyclic photophosphorylation was limited by photosystem II-mediated electron flow. Photosystem I-driven electron transport was substantially more stable over a wide pH range.     

ATP-driven proton fluxes across membranes of secretory organelles

The ATP-dependent proton uptake by chromaffin granule membranes, lysosomes, and synaptosomes was examined. In synaptosomes the reaction was absolutely dependent on the presence of chloride, while in chromaffin granules chloride had a profound effect and in lysosomes only a minor effect. The presence of chloride markedly increases the rate of collapse of delta pH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in all three organelles. Ascorbate with phenazine methosulfate uncoupled the ATP-dependent proton uptake by chromaffin granules, but had no effect on lysosomes and synaptosomes. Proton uptake by submitochondrial particles was about 50-fold more sensitive to dicyclohexylcarbodiimide than the proton uptake by chromaffin granule membranes. Chromaffin granule membranes were treated with 2 M sodium bromide to inactivate the mitochondrial ATPase. The treatment caused a complete inhibition of the ATP-dependent proton uptake. Solubilization of these membranes by sodium cholate, followed by reconstitution by cholate dilution revealed the ATP-dependent proton uptake of the system. It is concluded that the genuine ATPase enzyme of chromaffin granules is a proton translocator.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

The relation of the alteration of photosystem. II. Activity to the inhibition of energy transfer and the proton pump in tris-washed chloroplasts

Washing of spinach chloroplasts with high concentrations of Tris³ induces pH-dependent changes in chloroplast reactions. At high pH (8.4) Tris washing causes the inhibition of Photosystem 2 activity which can be prevented by the maintenance of reducing conditions during washing. Washing at low pH (7.2) causes an enhancement of oxygen evolution and increased rate of ferricyanide photoreduction which is not influenced by the presence of reducing conditions. The increased rate of electron flow is accompanied by the inhibition of light mediated phosphorylating activity, acid-induced ATP synthesis, light-induced proton uptake and light triggered Mg²⁺ ATPase activity. Tris treatment at low pH also causes a sensitization of Photosystem 2 activity such that oxygen evolution is inhibited by low concentrations of tris at high pH. This inhibition of the stimulated electron flow is not accompanied by a reconstitution of the photophosphorylation activity. A detailed analysis of the effect of tris treatment on Photosystem II activity and membrane dependent energy conversion shows that the treatment of chloroplasts causes an inhibition of the energy conversion process which is independent of the effect on oxygen evolution. Determination of the presence of coupling factor (as determined by ATPase activity) and membrane osmotic properties reveal normal levels of enzyme activity and osmotic response in treated chloroplasts. The inhibition of the energy conversion process is accompanied by reduced capacity to maintain a proton gradient. Kinetic analysis of the proton uptake reaction reveals that Tris treatment renders the grana membranes more permeable to protons.     

Photophosphorylation and related properties of reaggregated vesicles from spinach photosystem I particles.

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH+4 are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Photophosphorylation and related properties of reaggregated vesicles from spinach Photosystem I particles

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylalation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH⁺₄ are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Analysis of light-induced proton uptake in isolated chloroplasts

1. 1. A simple kinetic analysis of light-induced proton uptake into chloroplasts is presented. It is derived from a model of the reaction in which the incoming proton is obligatorily bound by an intra-chloroplast component, and allows quantitative analysis of the effect into parameters of light and dark rate constants and the availability of the chloroplast component.

2. 2. The effect of the following agents on the derived parameters has been measured: electron and energy transfer inhibitors, uncouplers, NaCl concentration, light intensity and pH.

3. 3. A maximal ratio of 4 protons taken up per electron transported has been observed, using ferricyanide as an electron acceptor.

4. 4. A stimulation of light-induced proton uptake by phosphate or arsenate, ADP and Mg has been observed. It was not sensitive to concentrations of Dio-9, which eliminated ATP synthesis.

5. 5. The results are seen as inconsistent with the chemiosmotic theory of energy coupling as presently presented. It is suggested that they may be interpreted in terms of a model in which the function of the proton pump is to enable co-transport into the chloroplasts of the negatively charged complex of phosphate, ADP and Mg.

Inside-out membrane vesicles isolated from spinach thylakoids.

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803.

cotA of Synechocystis sp. strain PCC6803 was isolated as a gene that complemented a mutant defective in CO2 transport and is homologous to cemA that encodes a chloroplast envelope membrane protein (A. Katoh, K.S. Lee, H. Fukuzawa, K. Ohyama, and T. Ogawa, Proc. Natl. Acad. Sci. USA 93:4006-4010, 1996). A mutant (M29) constructed by replacing cotA in the wild-type (WT) Synechocystis strain with the omega fragment was unable to grow in BG11 medium (approximately 17 mM Na+) at pH 6.4 or at any pH in a low-sodium medium (100 microM Na+) under aeration with 3% (vol/vol) CO2 in air. The WT cells grew well in the pH range between 6.4 and 8.5 in BG11 medium but only at alkaline pH in the low-sodium medium. Illumination of the WT cells resulted in an extrusion followed by an uptake of protons. In contrast, only proton uptake was observed for the M29 mutant in the light without proton extrusion. There was no difference in sodium uptake activity between the WT and mutant. The mutant still possessed 51% of the WT CO2 transport activity in the presence of 15 mM NaCl. On the basis of these results we concluded that cotA has a role in light-induced proton extrusion and that the inhibition of CO2 transport in the M29 mutant is a secondary effect of the inhibition of proton extrusion.     

Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.     

Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP-synthase

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through mitochondrial coupling factor 6-depleted F0 complexes

Submitochondrial particles prepared by treatment of mitochondria with ammonia and silicotungstic acid were found to be deficient in coupling factor 6 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting and had reduced ATP-Pi exchange activity. Requirement of coupling factor 6 for passive proton conductance through mitochondrial F0 was investigated by assaying the ability of depleted particles to sustain NADH-induced proton fluxes as measured by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The depleted particles themselves showed negligible quenching, but the quenching increased markedly after treating the particles with oligomycin. The data show for the first time that coupling factor 6-depleted complexes have an active proton channel that can be blocked by oligomycin. Therefore, coupling factor 6 is not essential for inhibitor-sensitive proton conductance through mitochondrial F0.     

The reversibility of freeze/thaw injury to spinach thylakoids; restoration of light-induced proton pumping,membrane-conformational changes and proton gradient formation

Spinach thylakoids have been frozen under a variety of salt / sucrose concentrations to remove varying amounts of peripheral membrane proteins, including the water-soluble part of the coupling factor complex (CF₁). This leads to a defined degree of uncoupling by exposing the CF₀ proton channel. The ability of thylakoids, subjected to this treatment, to reconstitute light-induced proton pumping, membrane-conformational changes and proton gradient formation when treated with DCCD, an energy transfer inhibitor which interacts with the CF₀ proton channel, thus reducing the proton permeability of the membrane, has been investigated. Full reconstitution of proton pumping and ΔpH formation could be obtained in thylakoids in which up to 75% of the coupling factor complex had been removed by the freezing regime. Even under the most severe conditions employed, in which over 80% of the CF₁ was removed from the membrane, there was still between 25 and 50% reconstitution of proton pumping. Reconstitution of membrane conformational changes as monitored by 90° scattering changes showed a strong positive correlation to the restoration of proton pumping. Reconstitution of slower, light-induced transmittance changes, in contrast, exhibited a more variable response. Little reconstitution of the slow transmittance changes was found under conditions which removed more than 60–70% of the coupling factor complex.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Effect of pyridine homologues on the basal rate of electron transport and H+/e − ratio in chloroplasts

Low concentrations of hydrophobic pyridine homologues (1 mM) were found to increase the rate of the Hill reaction in chloroplasts without significantly affecting either the steady-state proton uptake or the rate of proton leakage in the dark. By assuming that the organic base can be bound to two types of independent binding sites in the thylakoid membrane with dissociation constantsK ₁ andK ₂ respectively, the kinetic data can be treated quantitatively. The values ofK ₁ andK ₂ determined by the treatment are in the same relative order as the hydrophobicities of the pyridine homologues:K ₁=1.16 mM andK ₂=54 mM for pyridine; 0.6 and 38 mM for 4-picoline; 0.27 and 31 mM for 4-ethylpyridine, 0.10 and 4.2 mM for 4-t-butylpyridine; 0.08 and 3.2 mM for 4-n-butylpyridine. The rates of oxygen generation and proton uptake by illuminated chloroplasts with either ferricyanide or 1,4-benzoquinone as the electron acceptor were also measured in the presence of various pyridine homologues. Low concentration of pyridine homologues were found to decrease the H⁺/e ^– ratio. This last observation seems to substantiate an indirect coupling mechanism between electron transport and proton translocation.Abbreviations Chl chlorophyll - CF₀ - CF₁ the coupling factor complex of chloroplast - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Tricine N-tris-(hydroxymethyl)methylglycine     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F1-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose/2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF1-ATPase. The molecular weights of pea mitochondrial F1-ATPase and pea chloroplast CF1-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F1-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF1-ATPase. The purified mitochondrial F1-ATPase exhibited coupling factor activity. In spite of the observed differences between CF1 and F1, the mitochondrial enzyme stimulated ATP formation in CF1-depleted pea chloroplast membranes. Thus, the mitochondrial F1 was able to substitute functionally for the chloroplast CF1 in reconstituting photophosphorylation.     

IDENTIFICATION OF CHLOROPLAST COUPLING FACTOR BY FREEZE-ETCHING AND NEGATIVE-STAINING TECHNIQUES

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.     

Alterations in Chloroplast Thylakoids during an in Vitro Freeze-Thaw Cycle

Plastocyanin and chloroplast coupling factor 1 (CF(1)) are released from spinach (Spinacia oleracea L.) thylakoids during a slow freezethaw cycle. CF(1) addition increases the proton uptake of thylakoids previously frozen in sucrose concentrations of 15 mm to 100 mm. Addition of CF(1) and plastocyanin restores the proton uptake of thylakoids frozen in 100 mm sucrose. Plastocyanin and CF(1) release is a manifestation, not the cause, of freeze-thaw damage.Frozen-thawed thylakoids appear to exhibit two levels of response to sucrose as measured by light-dependent proton uptake. Different levels of protection afforded by sucrose may be due, in part, to quantitative differences in CF(1) release. The results suggest at least three freeze-induced lesions in light-dependent proton uptake by thylakoids: plastocyanin release, CF(1) release, and disruption of the semi-permeability of thylakoids.     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O₂ evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.