Effect of Mg2+ depletion of mitochondria on their permeability to K+: the mechanism by which ionophore A23187 increases K+ permeability.

Ionophore A23187 produces rapid swelling of rat liver mitochondria suspended in isotonio KNO₃ if an uncoupler and EDTA are also present. It also produces swelling of mitochondria in isotonic Mg(NO₃)₂ in the presence of an uncoupler. Washing with serum albumin removes the ionophore from mitochondria, as indicated by lack of swelling in magnesium nitrate (+ uncoupler). However, such treatment does not abolish rapid swelling in KNO₃ (+ uncoupler). This finding is interpreted in the sense that depletion of mitochondrial magnesium mobilizes K⁺/H⁺ antiport in the inner mitochondrial membrane.     

Proton transfer through the membrane—water interfaces in uncoupled mitochondria

Increase in maximal respiration rate of uncoupled mitochondria in response to increase in concentration of non-penetrating buffer has been demonstrated. This phenomenon did not depend on chemical structure of uncouplers and composition of the non penetrating buffer. Use of covalently attached pH probe, FITC, revealed that at low buffer concentration (3 mM) the H⁺-pump functioning results in local increase in proton concentration on the outer surface of the inner mitochondrial membranes. In other words, local H⁺ gradient was generated. Increase in buffer concentration up to 20 mM caused sharp decrease in this gradient, which occurred in parallel to increase in the respiration rate. It is concluded that both effects described here may be attributed to the process of proton transfer through the interfaces of the mitochondrial membrane: the rate of respiratory H⁺ pumps of uncoupled mitochondria under conditions of low buffer capacity of medium is limited by the stage of proton release from outer surface of the coupling membrane. The inhibition mechanism of respiration by high concentrations of uncouplers is also discussed.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 240–245.Original Russian Text Copyright © 2005 by Yurkov, Fadeeva, Yaguzhinsky.This revised version was published online in April 2005 with corrections to the post codes.     

Interaction of a surface-active base with the fraction of membrane-bound Williams’ protons

According to the Williams model, the work of mitochondrial respiratory H⁺ pumps gives rise to a fraction of membrane-bound protons (R-protons) that have excess free energy, which is used in the reaction of ATP synthesis. We have earlier managed to detect such a fraction in mitochondria and mitoplasts and to rigorously show (for mitoplasts) that the non-equilibrium R-proton fraction is localized on the surface of the inner membrane. Here we show that a surface-active compound 2,4,6-trichloro-3-pentadecylphenol anion (TCP-C₁₅) selectively interacts with the R-proton fraction, and describe in detail its influence on mitochondrial respiration under conditions of R-proton generation. We also report endogenous regulation of the R-proton fraction volume, which is performed by the phosphate transport system. The results are discussed in terms of the local coupling model.     

Redox dependence of transport activity of tonoplast proton pumps: Effects of nitric oxide exposure during ontogenesis and under hypoosmotic and hyperosmotic stress

Variations of the redox status is shown to inhibit the transport activity of tonoplast proton pumps at different stages of ontogenesis and under the conditions of hyperosmotic stress. However, the activity of H⁺-ATPase increased by 60% under hypoosmotic stress in the presence of GSH. The influence of nitric oxide on the transport activity of tonoplast proton pumps also depended on the redox status. In the case of change of the redox status, stimulating effect of nitric oxide turned inhibitory, except for simultaneous application of hypoosmotic stress and nitric oxide. In this case, stimulation of both proton pumps was observed and the activity of H⁺-ATPase increased in the presence of GSH, though the activity of H⁺-PPase increased in the presence of GSSG. This may explain the necessity of the presence in the vacuolar membrane of two proton pumps having similar functions.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Characterization of a chloroplast inner envelope P-ATPase proton pump

P-ATPases such as the plasma membrane proton pump are known to generate a phosphorylated intermediate as a step in their reaction mechanism; phosphoenzyme formation is a basis for classification of an ATPase as a member of this subfamily of ion pumps. The chloroplast inner envelope is known to contain a H+-ATPase which acts to maintain an alkaline stroma and, thus, optimal photosynthesis. Our characterization of this chloroplast envelope proton pump described in this report focused on determining whether purified chloroplast inner envelope membrane protein preparations containing this ATPase form a phosphorylated intermediate. Incubation of envelope membranes with [-³²P]ATP documented the formation of P-type ATPase phosphoenzyme intermediates by these membrane protein preparations. Our work cannot discount the possibility that more than one chloroplast inner envelope ATPase contributes to this phosphoenzyme formation. However, the kinetics of this phosphoenzyme formation, along with the sensitivity of phosphoenzyme formation to inhibitors and other assay conditions suggested that one of the envelope membrane proteins which is covalently radiolabeled by [-³²P]ATP is a P-type H+-ATPase. Autoradiography of chloroplast envelope membrane proteins size fractionated on lithium dodecyl sulfate-PAGE indicated that the phosphoenzyme intermediate corresponds to a 103 kDa polypeptide. P-type proton pumps are known to be comprised of a single type of 100 kDa subunit. Experimental evidence presented in this report is consistent with the classification of a chloroplast inner envelope H+-ATPase as a P-type proton pump.     

Alteration of transport activity of proton pumps in coleoptile cells during early development stages of maize seedlings

Comparative analysis of the transport activity of proton pumps (plasmalemma H⁺-ATPase, vacuolar H⁺-ATPase, and vacuolar H⁺-pyrophosphatase) in the membrane preparations obtained from coleoptile cells of etiolated maize seedlings (Zea mays L.) was carried out. The highest level of vacuolar pyrophosphatase activity was observed during the early development of coleoptile cells under growth intensification through the elongation. The role of ATPase pumps of tonoplast and plasmalemma in the transport of hydrogen ions increases during further development. The plasmalemma activity in this process is higher. When the growth stops, the activity of proton pumps becomes significantly lower. Nevertheless, their substrate specificity and sensitivity to proton pump inhibitors do not change, which can be an evidence of physiological significance of pumps in the maintenance of cell homeostasis.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Respiration-driven proton translocation in rat liver mitochondria

1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H⁺ (or OH⁻) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the →H⁺/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H⁺/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the →H⁺/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na⁺. These apparent changes of →H⁺/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H⁺/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H⁺/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.     

Generation of a proton gradient in Desulfovibrio vulgaris

Washed cells of Desulfovibrio vulgaris strain Marburg oxidized H₂, formate, lactate or pyruvate with sulfate, sulfite, trithionate, thiosulfate or oxygen as electron acceptor. CuCl₂ as an inhibitor of periplasmic hydrogenase inhibited H₂ and formate oxidation with sulfur compounds, and lactate oxidation in H₂-grown, but not in lactate-grown cells. H₂ oxidation was sensitive to O₂ concentrations above 2% saturation. Carbon monoxide inhibited the oxidation of all substrates tested. Additions of micromolar H₂ pulses to cells incubated in KCl in the presence of various sulfur compounds (reductant pulse method) resulted in a reversible acidification. This proton release was stimulated by thiocyanate, methyl triphenylphosphonium (MTPP⁺) or valinomycin plus EDTA, and completely inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), CuCl₂ or carbon monoxide. The extrapolated H⁺/H₂ ratios obtained with sulfate, sulfite, trithionate or thiosulfate varied from 1.0 to 1.7. Micromolar additions of O₂ to cells incubated in the presence of excess of electron donor (oxidant pulse method) caused proton translocation with extrapolated H⁺/H₂ ratios of 3.9 with H₂, 1.6 with lactate and 2.4 with pyruvate. Since a periplasmic hydrogenase can release at maximum 2 H⁺/H₂, it is concluded that D. vulgaris is able to generate a proton gradient by vectorial proton translocation across the cytoplasmic membrane and by extracellular proton release by a periplasmic hydrogenase.     

Membrane tubulation and proton pumps

Summary A model is presented for one type of membrane tubulation. In this model large transmembrane protein complexes link together to form helical bands. To form the helical bands the individual complexes within the band would need to form a slight bend and twist with adjacent complexes as they link together. Such linkages would lead to the band and lipid bilayer extending out from the plane of the membrane to form a tube of a diameter equal to or smaller than the radial diameter of the helix. This model is supported by two examples of membrane tubulation found inParamecium, in which proton pumps are the transmembrane complexes that are associated together to form the helically coiled bands. These include the helically linked F₀F₁ complexes of the mitochondrial inner membrane and the V₀V₁ complexes of the decorated tubules of the contractile vacuole complex. Such tubules result in very high surface-to-volume ratios and may be important in efficient ATP production or in forming proton gradients for transporting ions across membranes.     

Effects of Cr on proton extrusion, potassium uptake and transmembrane electric potential in maize root segments

Abstract Proton extrusion of maize root Zea mays segments, was inhibited by the presence of Cr (o.n. + 6; present in solution as CrO₄^2-, Cr₂O₇^2-) in the incubation medium: the minimum inhibiting concentration was 2 × 10^?3 mol m^?3 and the inhibition progressively increased with Cr concentration. Cr inhibited proton extrusion. Also, when this activity was stimulated by the presence of K⁺ or fusicoccin (FC) in the incubation medium, the K⁺ and FC stimulating effect was still present when proton extrusion was inhibited by Cr. In addition, Cr inhibited K⁺ uptake. This inhibition was higher (50%) at K⁺ concentrations up to 1 mol m^?3 lower (15%) at higher K⁺ concentrations. This result indicates that the system responsible for K⁺ uptake operating at low K⁺ concentrations is more sensitive to Cr inhibition. Cr had no effect on transmembrane electric potential (PD). The depolarizing and hyper-polarizing effect of K+ and FC, respectively, were not affected by Cr; but Cr enhances the depolarizing effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCP). These results indicate that Cr inhibited the proton translocating mechanism coupled with K⁺ uptake, but did not change the net transport of charges through the plasmalemma. The Cr effect is discussed, taking into account the possibility of a direct effect of Cr at the membrane level or, alternatively, of an effect on some metabolic processes controlling membrane function.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

A study of dependence of protein synthesis in mitochondria on the transmembrane potential

Summary 1. Incorporation of [H³]leucine into the TCA insoluble fraction of rat liver mitochondria incubatedin vitro is inhibited by uncouplers of oxidative phosphorylation. The inhibition is not correlated with the activation of mitochondrial ATPase. 2. Dependence of mitochondrial protein synthesis on the transmembrane potential is manifested in a wide range of K⁺ and Mg⁺⁺ concentrations in the incubation media. 3. The inhibitory action of uncouplers shows a lag period equal to 5–7 minutes, this lag period however is not observed when the uncoupler is added to puromycin-treated mitochondria. 4. Dependence of mitochondrial protein synthesis on the transmembrane potential, which represents a property characteristic for the inner mitochondrial membrane suggests that mitochondrial ribosomes act in close contact with the mitochondrial membrane system.Abbreviations MPS mitochondrial protein synthesis - CAP chloramphenical - CCP 2,4,6-chlorocarbonyl cyanide phenylhydrazone - FCCP p-trifluoromethoxy carbonyl cyanide phenylhydrazone - PEP phosphoenolpyruvate - Pi inorganic phosphate     

NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na⁺ uptake, indicating that a redox-driven, primary Na⁺ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 μmol min⁻¹ mg⁻¹) to Na⁺ translocation (2.0 μmol min⁻¹ mg⁻¹). NADH-driven Na⁺ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na⁺ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na⁺ gradient established by complex I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The proton pumps of the plasmalemma and the tonoplast of higher plants

Studies on intact cells, membrane vesicles, and reconstituted proteoliposomes have demonstrated in higher plants the existence of an ATP-driven electrogenic proton pump operating at the plasmalemma. There is also evidence of a second ATP-driven H⁺ pump localized at the tonoplast. The characteristics of both these ATP-driven pumps closely correspond to those of the plasmalemma and tonoplast proton pumps ofNeurospora and yeasts.     

Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H⁺-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H⁺-transport. At saturating (3 millimolar) concentrations of Mg²⁺:ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H⁺-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO₃, unlike the ATP-dependent H⁺-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H⁺-translocating pyrophosphatases. Both potassium and a permanent anion (NO₃⁻ > Cl⁻), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N′-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.     

Effects of W-7, W-5, Verapamil and Diltiazem on vacuolar proton transport. Comparison of vacuolar H+-ATPase and H+-PPase from roots of Zea mays

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H⁺-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H⁺-ATPase and H⁺-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺-ATPase (H⁺-PPase) were: 71 (191) μM W-7, 470 (> 800) μM W-5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺-ATPase, n_H= 2.02; H⁺-PPase, n_n= 0.96). The data indicate that the vacuolar H⁺-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H⁺-ATPase.