On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Cation-induced increases in the rate of P700 recovery in photosystem I particles.

Divalent cations such as Ca²⁺ and Mg²⁺ ions increase the rate of the dark recovery of P700⁺ in the P700-chlorophyll a protein of Shiozawa et al. (J. A. Shiozawa, R. S. Alberte, and J. P. Thornber, 1974, Arch. Biochem. Biophys., 165, 388–397). Half-maximal increases were observed at 1.5 mm concentrations of Mg²⁺ and Ca²⁺ ions. This correlates very well with the concentrations required to cause conformational changes in the P700-chlorophyll a protein. Na⁺ and K⁺ ions were also effective but 16–22 mm concentrations were required for half-maximal effects. Addition of Triton X-100 at concentrations greater than 0.02% also increased the rate of the dark recovery of P700⁺. The increases in the rate of P700 recovery are caused by a structural change involving the disaggregation of the protein. Mg²⁺ ions increase the rate of recovery of P700⁺ when both negatively (ascorbate and dichlorophenol indophenol) and positively (tetramethyl phenylenediamine) charged electron donors are used. This rules out the possibility that cations simply change the net charge on the protein to increase the binding of negatively charged electron donors. Moreover, it appears that Mg²⁺ ions affect the electron transport step rather than the binding of the donors to the complex. In addition, Mg²⁺ ions affect only the linear electron transport process from donor to O₂, not the recombination of P700⁺ with the primary electron acceptor.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb⁺ (K⁺) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb⁺ (K⁺) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca²⁺ (Gardos effect). The electron donors require the presence of intracellular Ca²⁺ to be effective, but at levels that do not stimulate by themselves the fluxes of K⁺. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca²⁺ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca²⁺ more effective to elicit rapid K⁺ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Direct measurement of k channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers

Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of H⁺. This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg²⁺ current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K⁺ over Cl⁻. The data are consistent with a role for these channels in charge balance during light-driven H⁺ movements.     

Transport of organic anions through the erythrocyte membrane as K+-valinomycin complexes

Summary K⁺, Rb⁺, or Cs⁺ complexes of valinomycin form ion pair complexes with picric acid and trinitrobenzenesulfonate (TNBS). The formation of a picrate-K⁺-valinomycin complex is supported by spectral evidence. These complexes have zero net charge and readily permeate the intact erythrocyte membrane. The K⁺-valinomycin complex has been used to convert the nonpenetrating TNBS into a penetrating covalent probe, making it as useful vectorial probe to measure accessible amino groups of proteins and phospholipids on both sides of the erythrocyte membrane.The enhanced transport of TNBS into the cell by valinomycin is dependent on external K⁺ in the medium. The entry of TNBS into the cell is manifested by an increased labeling of hemoglobin and membrane phosphatidylethanolamine (PE).Stilbeneisothiocyanatedisulfonate (SITS) and anilinonaphthalenesulfonate (ANS) inhibit both the basal and K⁺-valinomycin stimulated labeling of PE and hemoglobin by TNBS. The data suggest two independent effects of ANS and SITS, one mediated by an inhibition of the anion transport protein and another by the incorporation of these hydrobic anions into the cell membrane with an increase in negative charge on the membrane which leads to an inhibition of TNBS permeation into the cell by electrostatic repulsion.     

Kinetic analysis of P-700 photoconversion: Effect of secondary electron donation and plastocyanin inhibition

The kinetics of P-700 photoconversion under weak continuous actinic illumination were quantitatively analyzed to provide information on the relative absorption cross-section σ_PSI of the light-harvesting pigments associated with photosystem I and on the number of electrons stored between the two photosystems in dark-adapted chloroplasts. The theory of chemical kinetics for a system of monomolecular consecutive first-order reactions is reviewed briefly to provide support for the experimental approach taken. A complete inhibition of plastocyanin by cyanide eliminated all secondary electron donation to P-700⁺ and allowed the registration of the exponential (monomolecular) P-700 photoconversion at room temperature. The rate constant K_p-700 of the exponential kinetics was independent of the ionic (± Mg²⁺) and osmotic (± sucrose) strength of the chloroplast suspension medium, and of the oxidation-reduction state of photosystem II. The extent of plastocyanin inhibition in partially inhibited samples was greater under low ionic and low osmotic conditions. In dark-adapted chloroplast samples that were not cyanide treated, the number of electrons stored between the two photosystems was 3.9 ± 0.2 and independent of divalent cations. It is concluded that plastocyanin inhibition by cyanide is favored under low ionic and low osmotic conditions. The Mg²⁺ ion and redox state of photosystem II-independent photoconversion of P-700 does not support significant changes in the spillover of excitation from photosystem II to photosystem I in isolated chloroplasts.     

Energetic performance is improved by specific activation of K fluxes through KCa channels in heart mitochondria

Mitochondrial volume regulation depends on K⁺ movement across the inner membrane and a mitochondrial Ca²⁺-dependent K⁺ channel (mitoK_Ca) reportedly contributes to mitochondrial K⁺ uniporter activity. Here we utilize a novel K_Ca channel activator, NS11021, to examine the role of mitoK_Ca in regulating mitochondrial function by measuring K⁺ flux, membrane potential (ΔΨ_m), light scattering, and respiration in guinea pig heart mitochondria. K⁺ uptake and the influence of anions were assessed in mitochondria loaded with the K⁺ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH₂PO₄) salts of K⁺ to energized mitochondria in a sucrose-based medium. K⁺ fluxes saturated at ∼ 10 mM for each salt, attaining maximal rates of 172 ± 17, 54 ± 2.4, and 33 ± 3.8 nmol K⁺/min/mg in KCl, KAc, or KH₂PO₄, respectively. NS11021 (50 nM) increased the maximal K⁺ uptake rate by 2.5-fold in the presence of KH₂PO₄ or KAc and increased mitochondrial volume, with little effect on ΔΨ_m. In KCl, NS11021 increased K⁺ uptake by only 30% and did not increase volume. The effects of NS11021 on K⁺ uptake were inhibited by the K_Ca toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH₂PO₄, but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized ΔΨ_m. A control compound lacking K_Ca activator properties did not increase K⁺ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K⁺ flux through mitoK_Ca mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨ_m.     

Plastoquinol diffusion in linear photosynthetic electron transport

The diffusion of plastoquinol and its binding to the cytochrome bf complex, which occurs during linear photosynthetic electron transport and is analogous to reaction sequences found in most energy-converting membranes, has been studied in intact thylakoid membranes. The flash-induced electron transfer between the laterally separated photosystems II and photosystems I was measured by following the sigmoidal reduction kinetics of P-700⁺ after previous oxidation of the intersystem electron carriers. The amount of flash-induced plastoquinol produced at photosystem II was (a) reduced by inhibition with dichlorophenyl-dimethylurea and (b) increased by giving a second saturating flash. These signals were simulated by a new model which combines a deterministic simulation of reaction kinetics with a Monte Carlo approach to the diffusion of plastoquinol, taking into account the known structural features of the thylakoid membrane. The plastoquinol molecules were assumed to be oxidized by either a diffusion-limited or a nondiffusion-limited step in a collisional mechanism or after binding to the cytochrome bf complex. The model was able to account for the experimental observations with a nondiffusion-limited collisional mechanism or with a binding mechanism, giving minimum values for the diffusion coefficient of plastoquinol of 2 × 10^-8 cm²s^-1 and 3 × 10^-7 cm²s^-1, respectively.     

Regulation of sodium transport in erythrocytes

The kinetic response of swine erythrocyte (Na + K)-ATPase to Na⁺ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na⁺. The dependence of ouabain-inhibited efflux of Na⁺ on internal Na⁺ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na⁺ was sigmoidal (n = 2.2) in cells containing high K⁺ but hyperbolic in preparations in which most of the intracellular K⁺ was replaced by Li⁺, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K⁺ is an allosteric (feedback) inhibitor of Na⁺ efflux and there are three Na⁺ sites which interact cooperatively.     

Effect of NaCl and KCl salts on the growth and solute accumulation of the halophyte Atriplex nummularia

Atriplex nummularia plants are able to grow well in the absence of significant amounts of Na⁺. Medium levels of salinity (100 mM NaCl or KCl) did not cause substantial inhibition of growth but increasing concentrations of salt induced a progressive decline in length and weight of the plants. This inhibition was significantly higher in KCl grown plants than in NaCl grown plants. In addition, although it has been proposed that both K⁺ and Na⁺ are involved in the osmotic adjustment of plants in response to high soil salinity, we show that Na⁺ ions contribute more efficiently than K⁺ ions to perform this function. Our results also indicate that most of the osmotic adjustment of the plant was due to the accumulation of inorganic ions. The strong inhibition of Rb⁺ transport caused by internal sodium suggests that this cation could be efficiently used by the plant and, as a consequence, the transport of other monovalent cations is down-regulated.     

Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center ‘X’ and correlation with optical signal ‘A2’

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700⁺ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center ‘X’, characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700⁺ A⁻₂ reaction pair and the ESR P-700⁺ center X⁻ reaction pair to indicate that signal A₂ and center X represent the same iron-sulfur center in Photosystem I.     

Correlation between thylakoid stacking and cation-induced decrease in photosystem I electron transport at saturating light intensity

A Mg²⁺-induced decrease of the rate of photosystem I (PS I) electron transport (DCIPH₂ → methyl viologen) in thylakoids under saturated light intensities has been reported earlier (S. Bose, J. E. Mullet, G. E. Hoch, and C. J. Arntzen, 1981, Photobiochem. Photobiophys.2, 45–52). A similar effect is observed with Na⁺, although the concentration required for half-maximal inhibition was higher by about two orders of magnitude. The cation effect was gradually abolished as the thylakoids were aged by incubation at 30 °C for 6 h. The loss of cation effect on PS I electron transport rate during aging was parallel to the corresponding loss of cation effect on thylakoid stacking. The cation concentration required for thylakoid stacking and the degree of inhibition as a function of cation concentration correlated strongly with the degree of thylakoid stacking. These observations indicated that the inhibition of the rate of PS I electron transport by cations is a consequence of cation-induced stacking of thylakoid membranes. The observed inhibition of the rate of PS I electron transport is discussed in terms of two hypotheses: (i) a fraction (20–30%) of the PS I complexes is trapped in the appressed region of grana and becomes unavailable to the electron donor (DCIPH₂) and (ii) the membrane structure is altered by the cations in such a manner that the rate constant of electron donation by the donor to the electron transport chain in the thylakoid is decreased.     

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.     

Proton Fluxes as a Response to External Salinity in Wild Type and NaCl-Adapted Nicotiana Cell Lines

Addition of 100 millimolar KCl, NaCl, or Na₂SO₄ strongly promoted acidification of the medium by cells of Nicotiana tabacum/gossii in suspension culture. Acidification was greater in the case of NaCl-adapted than in that of wild type cells, and strikingly so in KCl medium when fusicoccin (FC) was present. Back-titration indicated that net proton secretion in KCl medium was increased 4-fold by FC treatment in the case of adapted cells; but was not even doubled in wild type cells. Membrane potential was higher in NaCl-adapted cells. FC treatment hyperpolarized wild, but not NaCl-adapted cells, suggesting a higher degree of coupling between H⁺ efflux and K⁺ influx in adapted cells; FC enhanced net K⁺ uptake in adapted but not in wild cells. Acidification by cells suspended in 10 millimolar KCl was highly sensitive to vanadate, but that after addition of 100 millimolar KCl or NaCl was much less sensitive. Addition of 100 millimolar NaCl to wild type cells already provided with 10 millimolar KCl briefly accelerated, then slowed down the rate of acidification. If the addition was made after acidification had already ceased, alkalization was observed, particularly in the presence of FC. The results are consistent with the operation of a Na⁺-H⁺ antiporter.     

Electron transport control in chloroplasts. Effects of photosynthetic control monitored by the intrathylakoid pH

(1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pH_in). A decrease in pH_in due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg²⁺-ADP with P_i); Mg²⁺-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700⁺ reduction. In chloroplasts, without adding Mg²⁺-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 ($\text{Δ}\text{pH}\text{? 0.5–1.0}$). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700⁺ reduction in the presence of Mg²⁺-ADP (with P_i) and without adding Mg²⁺-ADP is dependent on external pH variations, showing a maximum value of $\text{K ? 3.5}$ at pH_out 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg²⁺-ADP and P_i.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Mechanisms of passive potassium influx in corn mitochondria

Corn mitochondria in 100 millimolar KCl show accelerated passive swelling upon addition of uncoupler. This unusual response has been compared with swelling produced by valinomycin, tripropyltin, and nigericin. It is concluded that the driving force for swelling lies with the chloride gradient and a high P_Cl:P_K ratio, the chloride influx creating a negative membrane potential. The action of uncoupler is to facilitate K⁺ influx via the endogenous H⁺/K⁺ antiporter. The antiporter is active over the pH range 6 to 8, is not sensitive to Mg²⁺ concentration, and is not inactivated by aging. It is not clear why corn mitochondria show this exceptional activity of the H⁺/K⁺ antiporter in K⁺ influx. It is speculated that during isolation the antiporter may be exposed or activated, and that it contributes to cyclic K⁺ transport and high State 4 respiration rates.     

Potassium Channels in Chara corallina: CONTROL AND INTERACTION WITH THE ELECTROGENIC H PUMP

Plasmalemma electrical properties were used to investigate K⁺ transport and its control in internodal cells of Chara corallina Klein ex Willd., em R.D.W. Cell exposure to solutions containing 10 mm KCl caused the potential, normally −250 millivolts (average), to depolarize in two steps. The first step was a 21 millivolt depolarization that lasted from 1 to 40 minutes. The second step started with an action potential and left the membrane potential at −91 millivolts, with a 10-fold reduction in resistance. We suggest that the second step was caused by the opening of K⁺ -channels in the membrane. This lowered the resistance and provided a current pathway that partially short-circuited the electrogenic pump. Although largely short-circuited, the electrogenic pump was still operating as indicated by: (a) the depolarized potential of −91 millivolts was more negative than Ek (=−42 millivolts in 10 mm K⁺); (b) a large net K⁺ uptake occurred while the cell was depolarized; (c) both the electrogenic pump inhibitor, diethylstilbestrol, and the sulfhydryl-reagent N-ethylmaleimide (which increased the passive membrane permeability) further depolarized the potential in 10 mm KCl.A two-phase recovery back to normal cell potentials occurred upon lowering the K⁺ concentration from 10 to 0.2 mm. The first phase was an apparent Nernst potential response to the change in external K⁺ concentration. The second phase was a sudden hyperpolarization accompanied by a large increase in membrane resistance. We attribute the second phase to the closing of K⁺ -channels and the removal of the associated short-circuiting effect on the electrogenic pump, thereby allowing the membrane to hyperpolarize. Further experiments indicated that the K⁺ -channel required Ca²⁺ for normal closure, but other ions could substitute, including: Na⁺, tetraethylammonium, and 2,4,6-triaminopyrimidine. Apparently, K⁺ -channel conductance is determined by competition between Ca²⁺ and K⁺ for a control (gating?) binding site.