CO2 reduction by intact chloroplasts under a diminished proton gradient.

9-Aminoacridine has been used to monitor the intrathylakoid pH of photosynthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO2 reduction. However, low concentrations of uncoupling amines such as NH3 or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO2-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) OR NIGERICIN, WHICH INHIBITED CO2 reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH4C1, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.     

Equilibration of the ATPase reaction of chloroplasts at transition from strong light to weak light

Broken chloroplasts activated by preillumination in the presence of dithiothreitol were supplied with phosphate and with a limited concentration of ADP. On re-illumination, ATP was formed until a steady state was attained. If after reaching the steady state light intensity was reduced to 20–50 W · m⁻², net ATP hydrolysis took place, but after some time in weak light the level of ATP re-increased. Similarly, a drop of transmembrane ΔpH followed by a slow recovery was observed. Further data indicate that the reversible changes of ATP level and ΔpH are the result of partial uncoupling induced by ATP during the preceding strong light period and of restoration of coupling within a few minutes in weak light. Since similar changes of endogenous ATP level were found when intact chloroplasts were subjected to a strong-light/weak-light transition, it is proposed that ATP-induced partial uncoupling may play a role in regulation of photosynthetic energy conservation as a means to dissipate abundant transmembrane electrochemical energy and to permit flexibility of the stoichiometry of ATP-to-NADPH production.     

CO2 reduction by intact chloroplasts under a diminished proton gradient

9-Aminoacridine has been used to monitor the intrathylakoid pH of photo-synthetically competent intact chloroplasts. Values obtained from 9-aminoacridine accumulation in the chloroplasts must be corrected for light-dependent binding of 9-aminoacridine to the thylakoid membranes. During nitrite reduction by intact chloroplasts, the intrathylakoid proton concentration increased. It decreased somewhat during CO₂ reduction. However, low concentrations of uncoupling amines such as NH₃ or cyclohexylamine, which rapidly penetrated the chloroplast envelope and decreased the intrathylakoid proton concentration, failed to reduce, and actually stimulated, rates of CO₂-dependent oxygen evolution even under rate-limiting light. In contrast, low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin, which inhibited CO₂ reduction, even appeared to increase the intrathylakoid proton concentration. As indicated by measurements of the 515 nm signal of the chloroplasts, the light-induced membrane potential was not much affected by low concentrations of the uncoupling amines, but was decreased by FCCP and by high concentrations of the amines. Even in the presence of high concentrations of NH₄Cl, ATP/ADP ratios of illuminated chloroplasts remained far above the ratios observed in the dark. In contrast, low concentrations of FCCP were sufficient to reduce ATP/ADP ratios to the dark value even under high intensity illumination. The observations are difficult to explain within the framework of the chemiosmotic hypothesis as presently discussed.     

Lack of ATP requirement for light stimulation of glycerate transport into intact isolated chloroplasts

Light increased the initial rate and the extent of glycerate uptake by intact isolated chloroplasts. Half-maximum stimulation occurred with 10 to 20 watts per square meter of red light. Preillumination of chloroplasts enhanced uptake in a subsequent dark period. The light effect was abolished by DCMU and also by uncoupling agents such as nigericin and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone.

Arsenate and phlorizin only inhibited glycerate uptake to the extent that metabolism in the chloroplast was decreased by insufficient ATP. The concentration of glycerate accumulated in the chloroplast stroma was not significantly decreased. Chloroplasts isolated from young pea shoots (Pisum sativum, L. cv Massey Gem) were depleted of ATP by incubation with inorganic pyrophosphate or with ATP analogs. These treatments also decreased metabolism of glycerate but the actual concentration of glycerate accumulated in the chloroplast stroma was not decreased.

The results indicate that glycerate uptake is driven by ion gradients established across the chloroplast envelope in the light. ATP is not involved in the transport of glycerate into chloroplasts, being required only for the subsequent metabolism of glycerate in the chloroplast stroma. It is proposed that glycerate transport may be coupled to the proton gradient established in the light across the chloroplast envelope.

     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels.From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness.

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels. From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.     

Transport of mono- and divalent cations across chloroplast membranes mediated by the ionophore A23187

Effects of the ionophore A23187 on isolated broken and intact chloroplasts in the pH range of 6.2 to 7.6 have been studied. In both types of chloroplasts, uncoupling of photosynthetic electron transport by A23187 (6–10 μm) was mediated either by Mg²⁺ or—in the absence of divalent cations (i.e., when EDTA was added to the medium)—by high concentrations of Na⁺, but not of K⁺ ions. At increased concentrations of the ionophore (above about 10 μm) and high pH (7.2 to 7.6), uncoupling in broken chloroplasts was also mediated by K⁺ ions. The inhibition of the energy-dependent slow decline of chlorophyll fluorescence in intact chloroplasts by the ionophore (which denotes uncoupling) is reversed by EDTA in the presence of K⁺, but not of Na⁺ ions. In 3-(3′,4′-dichlorophenyl)1,1-dimethylurea-poisoned intact chloroplasts, the yield of variable chlorophyll fluorescence is lowered by A23187 + EDTA and increased again by addition of NaCl or KCl. Chlorophyll fluorescence spectra at 77 °K of intact chloroplasts incubated with A23187 + EDTA indicated that the distribution of excitation energy had changed in favor of photosystem I, as expected from a depletion of Mg²⁺. This change was reversed by MgCl²⁺, KCl, or NaCl. From a comparison of low-temperature fluorescence spectra of broken and intact chloroplasts at different levels of Mg²⁺ in the medium, the concentration of free Mg²⁺ in the stroma of the intact chloroplasts at pH 7.6 in the dark was estimated at 1 to 4 mm. The results show that in chloroplasts the specificity of A23187 for divalent cations is limited. In the presence of EDTA, the ionophore mediates fast $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange across thylakoid membranes, whereas K⁺ is transferred much less efficiently. Both Na⁺ and K⁺ ions seem to be transported readily across the chloroplast envelope by the action of the ionophore, leading to an exchange of Mg²⁺ for monovalent cations at the thylakoid membrane surfaces in intact chloroplasts.     

Involvement of stromal ATP in the light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact isolated chloroplasts

Light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and stromal ATP content were measured in intact isolated spinach chloroplasts. Treatments which decreased stromal ATP, such as incubation with the ATP analog β,γ-methylene adenosine triphosphate or with the energy transfer inhibitor phloridzin inhibited the light activation of rubisco. In the absence of added inorganic phosphate (Pi), light activation of rubisco was inhibited, coincident with low stromal ATP. Addition of methyl viologen restored both stromal ATP and rubisco activity to levels observed in the presence of Pi. Activation of rubisco was inhibited in the presence of 2 millimolar dihydroxyacetone phosphate or 3-phosphoglycerate and stromal ATP was also decreased under these conditions. Both were partially restored by increasing the Pi concentration. The strong correlation between activation state of rubisco and stromal ATP concentration in intact chloroplasts under a wide variety of experimental conditions indicates that light activation of rubisco is dependent on ATP and proportional to the ATP concentration. These observations can be explained in terms of the rubisco activase protein, which mediates activation of rubisco at physiological concentrations of CO₂ and ribulose-1,5-bisphosphate and is dependent upon ATP.     

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion and amine uptake and uncoupling in submitochondrial particles.

1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KCl or NH4Cl. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-1-napthalene sulphonate cause a pH-independent, 50% uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 muM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two pHases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects; uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as Cl-, the uncoupling effect is independent of Cl-. Furthermore the amine uncoupling is markedly enhanced by lipophilic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Effects of Water Stress on Energy Transduction in Chloroplasts

Water stress inhibited the photosynthetic O2 evolution rate of wheat leaves. It was shown that water stress decreased the electron transport rate, the activities of photophosphorylation and, coupling factor, and, the synthesis of ATP in chloroplasts. PS Ⅱ electron transport was more senstitive to water stress than PS Ⅰ. The reduction in photophosphorylation activity might be the results of reduction in electron transport rate and coupling factor activity, as well as the uncoupling effect of water stress on chloroplasts. The uncoupling effect could be due to the inhibition of light induced proton translocation in chloroplasts.     

Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

Background  

Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration.     

Inhibition of the photosynthetic capacity of isolated chloroplasts by ozone

Isolated spinach chloroplasts have been used as a model system for studying the interaction of ozone, a component of photochemical smog, with plant membranes. Ozone bubbled into a suspension of isolated chloroplasts inhibits electron transport in both photosystems without uncoupling ATP production. Photosystem I (reduced 2,6-dichlorophenolindolphenol → NADP⁺) is a little more sensitive than photosystem II (H₂O → 2,6-dichlophenolindolphenol). Ozone does not act as an energy transfer inhibitor, since the drop in ATP production and high energy intermediate (measured by amine-induced swelling) is nearly parallel to the decline in electron transport. A reasonable hypothesis is that ozone disrupts the normal pathway of energy flow from light-excited chlorophyll into the photoacts by a disruption of the components of the membrane but not a general disintegration of the membrane. In addition, ozone does not seem to penetrate into the grana region through the outer membrane of intact plastids, since ozone lowers the bicarbonate-supported O₂ evolution but does not affect the rate of ferricyanide reduction in the same plastids after osmotic disruption. This would indicate that the effect of ozone on green plants, at low concentrations, may be due to the interaction of ozone with the first membrane it contacts and not directly with internal metabolic processes.     

Re-examination of the localization of Mg-chelatase within the chloroplast

In a plastid-free assay, Mg-chelatase from pea ( Pisum sativum L. cv. Spring) and cucumber ( Cucumis sativus L. cv. Sumter) chloroplasts is inhibited to equal extents by the mercurial reagents. p -chloromercuribenzoate (PCMB) and p -chloromercuribenzene sulfonate (PCMBS). However, in intact chloroplasts PCMB inhibits Mg-chelatase fourfold more strongly than does PCMBS. Since PCMBS cannot penetrate membranes as readily as PCMB, Mg-chelatase may be localized interior to the inner chloroplast envelope. When Mg-chelatase is assayed with photosynthetically generated ATP, the presence of an external ATP trap does not inhibit activity, suggesting that the enzyme is not located in the interenvelope space. None of the components of Mg-chelatase are integral membrane proteins: Mg-chelatase activity is readily solubilized by washing the total chloroplast membranes in buffers of low MgCl₂ content. This precludes localization by purifying individual thylakoid and envelope membranes which requires low MgCl₂ concentrations.     

ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis

In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol.     

Control of hydrogen-dependent nitrogenase activity by adenylates and electron flow in heterocysts of Anabaena variabilis (ATCC 29413)

Hydrogen-dependent nitrogenase activity was studied in heterocysts, isolated from the filamentous cyanobacterium Anabaena variabilis (ATCC 29413). Hydrogen provides reductant and ATP for nitrogenase via linear electron flow through Photosystem I. This allows for regulation of nitrogenase activity by controlling the turnover of the photosystem. When nitrogenase activity was varied by changing either the light intensity or the supply of reductant (i.e., hydrogen) or by inhibition of photosynthetic electron transport by DBMIB, no rate-dependent changes in cellular ATP concentrations were observed. This homeostasis of ATP was perturbed by addition of metronidazole, acting as alternative electron sink to nitrogenase, and by uncoupling agents like FCCP, gramicidin and nigericin. Valinomycin (in presence of KCl) exerted little effect on nitrogenase activity and adenylate pool composition. Metronidazole increased and uncoupling agents decreased cellular ATP concentration, ATP/ADP ratio and energy charge. Inhibition of nitrogenase activity by metronidazole was caused by reductant limitation; inhibition by uncoupling agents was due to energy limitation. Control exerted on nitrogenase activity by ATP (energy limitation) was more pronounced at high rates of electron flow to nitrogenase than during reductant limitation. When cellular ATP synthesis was suboptimal due to partial uncoupling, the connection of phosphorylation and nitrogenase activity by electron transport allowed for homeostasis of ATP also at a lowered cellular concentration.     

Localization of photophosphorylation and proton transport activities in various regions of the chloroplast lamellae

Membrane fragments released by French pressure cell treatment of whole chloroplasts and isolated by differential centrifugation have been characterized structurally and with respect to phosophorylating and proton transport activities. In agreement with results of other workers, the heavy fraction released by pressure treatment was found by electron microscopy studies to be made up of mostly intact grana stacks while the light fraction was comprised of vesicles derived from the stromal lamellae. Both fractions were found to carry out rapid rates of cyclic photophosphorylation catalyzed by phenazine methosulfate (PMS). However, only the grana membranes demonstrated active proton accumulation in the presence of PMS. No light induced H⁺ uptake could be detected in the stromal lamellae fraction; and as expected, proton gradient dissipating agents such as NH₄Cl, nigericin in the presence of K⁺, and gramicidin were only slightly inhibitory to phosphorylation at concentrations which were very inhibitory in the grana membrane fraction.

Further evidence that stromal lamellae do not have active proton transport in the intact chloroplast was obtained by comparing various chloroplasts having different amounts of stromal and grana membranes. Comparative studies on young and old chloroplasts from lettuce, mesophyll and bundle sheath cell plastids from sorghum, and greening plastids from etiolated corn seedlings revealed a direct correlation between the extent of grana formation and the amount of proton transport activity. Samples which had larger amounts of stromal lamellae had high rates of ATP formation but a reduced capacity for H⁺ accumulation.