Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Light energy conservation processes in Halobacterium halobium cells.

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

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells

In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Speculations on the evolution of ion transport mechanisms.

Primate cells evolved a plasma membrane to restrict the loss of important molecules. The osmotic problems that then arose were solved in one of several ways. Of major importance was the evolution of specific ion pumps, to actively extrude those salts whose inward diffusion would have led to swelling and lysis. In addition, these pumps allowed the cell to store energy in the form of ion gradients across the membrane. Thus, even in the earliest stages, the evolution of ion transport systems coincided with the development of mechanisms which catalyzes the energy transformations. It is postulated that an "ATP"-driven proton pump was one of the first ion transport systems. Such a proton pump would extrude hydrogen ions from the cell, establishing both a transmembrane pH gradient (alkaline inside) and a membrane potential (negative inside). This difference in electrochemical potential for protons (the proton-motive force) could then drive a variety of essential membrane functions, such as the active transport of ions and nutrients. A second major advance was the evolution of an ion transport system that converted light energy into a form which could be used by the cell. The modern model for this is the "purple membrane" of Halobacterium halobium, which catalyzes the extrusion of protons after the capture of light. The protonmotive force generated by such a light-driven proton pump could then power net synthesis of ATP by a reversal of the ATP-driven proton pump. A third important evolutionary step associated with ion transport was the development of a system to harness energy released by biological oxidations. Again, the solution of this problem was to conserve energy as a protonmotive force by coupling the activity of a respiratory chain to the extrusion of protons. Finally, with the development of animal cells a more careful regulation of internal and external pH was required. Thus, an ATP-driven Na+-K+ pump replaced the proton-translocating ATPase as the major ion pump found in plasma membranes.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

An alternative hypothesis for the direction of hydrogen ion movement and energy transduction in H. halobium.

It is generally accepted that purple membrane of $\text{H.}\text{halobium}$ functions as a light-driven hydrogen ion pump translocating hydrogen ions from inside the cell to the external medium. However, experimental data from this laboratory together with those obtained by others have always shown an initial alkalinization of the external medium in the light. Additionally, we have found that oxygen can also induce an alkalinization of the bathing solution in the dark. These results can be readily explained if the direction of hydrogen-ion translocation is reversed, that is that both light and oxygen generate an electrochemical gradient of hydrogen ions, which is outwardly directed for ATP synthesis.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Electrogenic transport by the Enterococcus hirae ATPase

A transport ATPase from Enterococcus hirae was reconstituted in lipid vesicles and its electrogenic action investigated with the fluorescent dye oxonol VI as membrane potential probe. Reconstitution in bacterial and in soybean phospholipid mixtures led to transport-active vesicle preparations. Inside-out oriented ATPase molecules were activated by the addition of ATP to the extravesicular medium, generating in all experiments an intravesicularly positive potential. The extravesicular pH strongly influenced the initial pumping rate and the duration of the pumping activity. At neutral pH, transient pumping activity was observed, lasting for 1-2 min, while at pH 5.6, pumping was continuous. The transport activity was not dependent on the ionic composition of the buffer on either side of the membrane. These findings can be interpreted as the action of a proton ATPase, regulated by the cytoplasmic proton concentration and electrogenically translocating protons from the cytoplasm to the extracellular space.     

Isotope and thermal effects in chemiosmotic coupling to the membrane ATPase of Streptococcus

We measured rates of ATP synthesis by the proton-translocating ATPase of the motile Streptococcus strain V4051. Starved cells were energized artificially by exposing their membranes to a variable electrical potential difference (internal medium negative) and a fixed pH difference (internal medium alkaline). The initial rates of ATP synthesis increased exponentially with protonmotive force. The results were the same in D2O and H2O; there was no solvent isotope effect. At a fixed protonmotive force, the rates were strongly dependent on temperature, as expected for a reaction with a large enthalpy of activation. At a different protonmotive force, the rates varied with temperature in an identical fashion; there was no change in the enthalpy of activation. We conclude that protonation-deprotonation steps are not rate limiting and that the protons that cross the membrane drive ATP synthesis by mass action. The transmembrane electric field acts by changing the concentrations of the reactants, not by changing the configuration of the enzyme-substrate complex.     

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity

Peribacteroid membrane vesicles from pea (Pisum sativum) root nodules were isolated from membrane-enclosed bacteroids by an osmotic shock. The ATPase activity associated with this membrane preparation was characterized, and its electrogenic properties were determined. The pH gradient was measured as a change of the fluorescence intensity of 9-amino-6-chloro-2-methoxyacridine and the membrane potential as a shift of absorbance of bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. It was demonstrated that the ATPase generates a pH gradient as well as a membrane potential across the peribacteroid membrane. The reversibility of the ATPase was demonstrated by a light-dependent ATP synthesis by peribacteroid membrane vesicles fused with bacteriorhodopsin-phospholipid vesicles. The light-driven ATP synthesis by the peribacteroid membrane ATPase was completely inhibited by a proton-conducting ionophore. The proton-pumping activity of the peribacteroid membrane ATPase could also be demonstrated with peribacteroid membrane-enclosed bacteroids, and effects on nitrogenase activity were established. At pH values below 7.5, an active peribacteroid membrane ATPase inhibited the nitrogenase activity of peribacteroid membrane-enclosed bacteroids. At pH values above 8, at which whole cell nitrogenase activity was inhibited, the protonpumping activity of the peribacteroid membrane ATPase could partially reverse the pH inhibition. Vanadate, an inhibitor of plasma membrane and peribacteroid membrane ATPases, stimulated nodular nitrogenase activity. It will be proposed that the proton-pumping activity of the peribacteroid membrane ATPase in situ is a possible regulator of nodular nitrogenase activity.     

ATP synthesis by ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716 by ionophore-induced electric potentials and proton gradients

ATP synthesis driven by low pre-established electric potentials and pH gradients is studied in large ATPase proteoliposomes, prepared from the ATPase complex and native lipids from the thermophilic cyanobacterium Synechococcus 6716. Electric potentials and pH gradients were achieved by valinomycin and nigericin, respectively, in the presence of a K⁺ gradient across the membrane. External base-pulses were also applied. In this system ATP synthesis driven by valinomycin-induced K⁺ influx, nigericin-induced internal acidification and by external base-pulses is demonstrated. Electric potentials and pH gradients of equivalent size lead to roughly similar ATP synthesis activities. ATP synthesis is optimal at 80–100 nM valinomycin and at 0.75−1 μM nigericin at the proper pre-set ion gradients. Uncoupler and DCCD inhibit ATP synthesis. Prior activation of the complex by thiol agents or trypsin was not required for synthesis activity. The ATP synthesis rate increases with the size of electric potential or pH gradient. The threshold value of the electrochemical gradient for significant ATP synthesis is about 30 mV. ATP production proceeds for more than 60 min. The generation of ionophore-induced electric potentials and pH gradients have been followed by oxonol VI and intraliposomal Neutral red, respectively. The extent of the absorbance changes of both probes is proportional to the size of electric potential or pH gradient. Ionophore-induced oxonol VI and Neutral red responses are stable for at least 30 min. The results are discussed in terms of membrane permeability and vesicle size.     

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.     

Increasing Acidification of Nonreplicating Lactococcus lactis ΔthyA Mutants by Incorporating ATPase Activity

Lactococcus lactis MBP71 ΔthyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.     

H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.     

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.     

Inhibition by triphenyltin chloride of a tightly-bound membrane component involved in photophosphorylation.

At very low concentrations (less than 1 muM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (-Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane-bount ATP in equilibrium Pi exchange and Mg2+-dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+-dependent and Mg2+-dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light-driven proton pump in chloroplasts is stimulated (up to 60%) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2-2.5 triphenyltin molecules/100 chlorophyll molecules at 50% inhibition of ATP formation and half-maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA was are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4-5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution. On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation.Atp in equilibrium Pi exchange and membrane-bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane-bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.     

Steady state kinetics of ATP synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor

The steady state kinetics of ATP synthesis and hydrolysis catalyzed by the chloroplast dicyclohexylcarbodiimide-sensitive ATPase reconstituted into phospholipid vesicles were studied as a function of the transmembrane proton gradient. Bacteriorhodopsin also was incorporated into the vesicles so that a constant pH gradient could be maintained by continuous illumination of the liposomes. The dependence of the initial rates of ATP synthesis and hydrolysis on substrate concentrations is consistent with Michaelis-Menten kinetics, with enzyme, ADP, and Pi forming a ternary complex. The Michaelis constants for both synthesis and hydrolysis are essentially independent of the pH gradient, while the maximum velocities depend strongly on it. The equilibrium constant for hydrolysis was calculated from the steady state kinetic parameters, and the dependence of the equilibrium constant on the pH gradient indicates that 3 protons are transported per ATP synthesized or hydrolyzed. The dependence of the steady state kinetic parameters on the pH gradient can be described by a mechanism in which the binding of substrates occurs before the transport of protons and the transport of the 3 protons is sequential rather than concerted.     

Proteoliposomes with right-side-out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation-free purple membranes carrying little activity of light-driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right-side-out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right-side-out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right-side-out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.     

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO₃ or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.