A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.     

The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

Proton-translocating adenosinetriphosphatase in rough and smooth microsomes from rat liver

Rat liver smooth and rough microsomal membranes exhibit an ATP-dependent H+ transport which can be inhibited by sulfhydryl reagents and dicyclohexylcarbodiimide but is resistant to oligomycin. On the basis of inhibitor sensitivities and substrate specificities, this H+ pump was found to be different from that of mitochondria, lysosomes, gastric H+-K+-ATPase, and yeast plasma membrane H+-ATPase but to resemble that of endocytic vesicles and the H+ pump responsible for urinary acidification. The transport process is accelerated by valinomycin in the presence of potassium, suggesting that it is an electrogenic pump. The same fractions were enriched in an ATPase with inhibitor sensitivities similar to those of the transport activity. It is possible that the proton electrochemical gradients generated by this pump may play a role in the translocation of proteins and sugars, two of the major functions of these structures.     

大豆液泡膜H~+-ATPase的纯化和重组

通过不连续蔗糖密度梯度离心得到的液泡膜微囊 ,先由胆酸钠和 OG分步破膜抽提、经阴离子交换柱 ( Q- Sepharose)层析分离 .纯化后的酶含 V型 H+ - ATPase的主要亚基 ,与大豆磷脂重组 ,获得了有较高泵活性的脂酶体 .脂酶体的质子泵活性受 Valinomycin激活 ,说明它是致电性的 ,受NO-3 ,DCCD以及特异性的 V型 ATPase抑制剂 Bafilomycin的抑制 .脂酶体的泵活性不受 F型和P型 ATPase抑制剂抑制 ,表明质子转运是由 V型 H+ - ATPase引起的 .     

Inhibition of clathrin-coated vesicle acidification by duramycin

Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.     

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.     

Plasma membrane proton ATPase from human kidney

Distal urinary acidification is thought to be mediated by a proton ATPase (H+-ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+-ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+-ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+-ATPase) and in the absence of Ca (to inhibit Ca2+-ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N-ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+-ATPase, respectively. Dicyclohexylcarbodiimide and N-ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward-directed K gradient, there was increased quenching of acridine orange, indicating that the H+-ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+-ATPase, which is similar to the H+-ATPase described in other species, and we postulate that this H+-ATPase may be involved in urinary acidification.     

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F₀ to F₁ ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N′-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.     

Aluminum inhibits the lysosomal proton pump from rat liver

Lysosomes are cytoplasmatic organelles, delimitated by a single lipoprotein membrane, that contain several enzymes mostly belonging to the hydrolases in that they function mainly for intracellular digestion. Lysosomal internal pH is characteristically acidic and it is maintained around pH 4.5 by a proton pump, an ATPase, that uses energy from ATP hydrolysis to translocate H+ ions into lysosomes. In the presence of Al3+ the proton pump activity is markedly reduced compromising acidic vesicles functionality. Among different species utilized, Al2(SO4)3 and AlF3 were the most effective. Aluminum effect was not observed when the delta pH was produced artificially by nigericin.     

Internal anion binding site and membrane potential dominate the regulation of proton pumping by the chromaffin granule ATPase

Effects of anions and membrane potential on the reconstituted proton pump from chromaffin granules were investigated. When acetate was present inside of the vesicles, ATP-dependent proton uptake was absolutely dependent on external chloride. Without external chloride, however, substantial proton uptake was observed when chloride or sulfate was present inside of the vesicles. Inside negative membrane potential drove ATP-dependent proton uptake regardless of the anion species present inside or outside of the vesicles. It is concluded that the internal anion binding site and membrane potential regulate the proton pumping activity of the ATPase.     

Role of Na+ cycle in cell volume regulation of Mycoplasma gallisepticum.

The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-s period and showed almost the same rate in the pH range of 6.5 to 8.0. 22Na+ efflux was markedly inhibited by dicyclohexylcarbodiimide (DCCD, 10 microM), but not by the proton-conducting ionophores SF6847 (0.5 microM) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) over the entire pH range tested. An ammonium diffusion potential and a pH gradient were created by diluting intact cells or sealed membrane vesicles of M. gallisepticum loaded with NH4Cl into a choline chloride solution. The imposed H+ gradient (inside acid) was not affected by the addition of either NaCl or KCl to the medium. Dissipation of the proton motive force by CCCP had no effect on the growth of M. gallisepticum in the pH range of 7.2 to 7.8 in an Na+-rich medium. Additionally, energized M. gallisepticum cells were stable in an isoosmotic NaCl solution, even in the presence of proton conductors, whereas nonenergized cells tended to swell and lyse. These results show that in M. gallisepticum Na+ movement was neither driven nor inhibited by the collapse of the electrochemical gradient of H+, suggesting that in this organism Na+ is extruded by an electrogenic primary Na+ pump rather than by an Na+-H+ exchange system energized by the proton motive force.     

The membrane potential of the cellular slime mold Dictyostelium discoideum is mainly generated by an electrogenic proton pump

Trans membrane potential or ionic current changes may play a role in signal transduction and differentiation in the cellular slime mold dictyostelium discoideum. Therefore, the contribution of electrogenic ion pumps to the membrane potential of D. discoideum cells was investigated. the (negative) peak-value of the rapid potential transient, seen upon microelectrode impalement, was used to detect membrane potential changes upon changes in the external pH in the range of 5.5 to 8.0. The membrane potential was close to the Nernstian potential for protons over the pH range 5.5 to 7.5. The acid-induced changes in membrane potential were consistent with outward-proton pumping. The maximal membrane potential was at pH 7.5. Furthermore, the proton pump inhibitors diethylstilbestrol, miconazole and zearalenone directly depolarize the membrane. Cyanide and temperature decrease cause membrane depolarization as well. During recovery from cyanide poisoning a H+ efflux is present. From these measurements we conclude that the membrane potential of d. discoideum cells is mainly generated by an electrogenic proton pump. Measurements in cells with different extracellular potassium and H+ concentrations suggest a role for potassium in the function of the electrogenic proton pump. These results provide a framework for future research towards a possible role for the proton pump in signal transduction and differentiation.     

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.     

Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells

Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ ATPase in LLC-PK1 cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1 cells were allowed to internalize cytochrome c-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ ATPase in these compartments, we labeled LLC-PK1 cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ ATPase immunoprecipitated from both fractions with monoclonal anti-H+ ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.     

Electrogenic nature of lysosomal proton pump as revealed with a cyanine dye

Studies were carried out on the electrogenicity of the lysosomal proton pump using dipropylthiadicarbocyanine iodide (diS-C3-(5] as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C3-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K+ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethylphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluorescein isothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATP-induced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate delta pH across the lysosomal membrane also enhanced the Mg-ATP-induced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K+ medium, mostly due to the K+-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [3H]TPMP and [14C]SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.     

Plasma membrane proton-ATPase of a turtle bladder epithelial cell line

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Electrogenicity of the lysosomal proton pump

Using acridine orange as a pH gradient probe, the effects of valinomycin and FCCP on the pH gradient across lysosomal membranes in an ATP-free medium as well as on the rate of the inward ATP-driven proton translocation were investigated. Both lysosome-enriched and highly purified lysosomal preparations from rat liver were used with identical results. Ionophore effects were found to be different depending upon whether passive ion fluxes or ATP-driven H+ translocation were involved and supported the existence of a membrane potential in the latter case. Anions stimulated the rate of ATP-driven proton translocation and stimulation increased with increasing anion lipophilicity. These results strongly support the electrogenic nature of the lysosomal proton pump.