ATP-dependent Proton Transport Research Articles

Characterization of ATP-Hydrolyzing and ATP-Driven proton-translocating activities associated with the peribacteroid membrane from root nodules of Lupinus luteus L

In order to detect and characterize PBM H + -ATPase from root nodules of Lupinus luteus L., ATP-hydrolyzing and ATP-dependent proton translocating activities associated with this membrane were studied using isolated symbiosomes, vesicle PBM preparations and intact plant tissues. It has been found that ATP-hydrolyzing activity of isolated PBMs is sufficiently high (about 100–150 μmoles of μmg protein-h) in the selected pH range (4.5–8.5) and is characterized by the absence of any pronounced pH optimum, little substrate specificity, the absence of selectivity in relation to Mg 2+ and Ca 2+ as stimulators of ATP-hydrolysis and even more sensitivity to Ca 2+ . Mg 2+ -dependent ATP-hydrolyzing activity was moderately decreased in the presence of some known inhibitors of H + -ATPases, such as vanadate, DCCD and nitrate. On the other hand, Ca 2+ -dependent ATP-hydrolysis was significandy inhibited by sodium fluoride. ATP-hydrolyzing activity on the PBM of Lupinus luteus L. with many similar properties was also detected cytochemically in electron microscope studies of intact plant tissues. These findings led us to conclude that at least one more Ca 2+ /Mg 2+ -dependent ATP-hydrolase, in addition to H + -ATPase, is associated with the PBM. This enzyme is likely responsible for the observed background that masks ATP-hydrolyzing activity of the PBM H + -ATPase. ATP-dependent electrogenic proton transport across the PBM was detected in isolated symbiosomes and vesicle PBM preparations using oxonol YI and acridine orange as indicators of ΔΨ and ΔpH, respectively. Transport activity of the PBM H + -ATPase was blocked by the above mentioned ATPase inhibitors, except nitrate, which acted only as permeant anion. These results indicate that the PBM of Lupinus luteus L. contains only one H + -translocating ATPase, which belongs to a P-type H + -ATPase.

Proton pump activation and growth promotion by cinchomeronic acid in radish

Cinchomeronic acid (CA, 3,4-dicarboxypyridine or 4-carboxynicotinic acid) promoted hypocotyl elongation of radish seedlings when added to the medium or applied to the hypocotyls. On microscopic observation of epidermal cells of the plant, the length of each cell was elongated several times when compared to those of the controls. The mechanism of the plant-growth promotion by CA was investigated with plasma membrane vesicle prepared from radish seedlings. CA elevated the Vmax of vanadate-sensitive H +-ATPase in plasma membrane vesicle by 1.8 times. It was found that CA and quinolinic acid were activators, and isocinchomeronic acid and 2,4-lutidinic acid were inhibitors for the H +-ATPase. These results reflected the effects of these compounds on plant growth. It is suggested that CA activates ATP-dependent proton transport and leads to plant-growth promotion by acid.

Regulation of Plasma Membrane V-ATPase Activity by Dissociation of Peripheral Subunits

The plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K+/nH+ antiport and thus K+ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K+ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V1 sector, whereas the membrane integral V0 subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V1 domain.

A V-ATPase drives active, electrogenic and Na+-independent Cl- absorption across the gills of Eriocheir sinensis

Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 50­60 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.

Activity and in vitro reassembly of the coated vesicle (H+)-ATPase requires the 50-kDa subunit of the clathrin assembly complex AP-2.

We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.

A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut.

A cDNA clone encoding a hydrophilic protein with a calculated molecular mass of 13,839 Da was isolated by shotgun screening with an anti-V-ATPase holoenzyme serum. The deduced amino acid sequence showed no significant homology to any other known protein. Southern blots revealed the existence of only one gene encoding the 14-kDa protein. Monospecific antibodies purified by affinity to the recombinant protein demonstrated the presence of a 14-kDa protein in the highly purified goblet cell apical membrane and inhibited ATP-dependent proton transport as well as V-ATPase activity to the same extent. Thus, the 14-kDa protein was shown to be a part of the V-ATPase holoenzyme. Binding of the monospecific antibodies to the ATPase seemed to require an ATP-dependent conformational change of the enzyme, since inhibition only occurred when ATP was present during the antibody binding step. The 14-kDa subunit could be stripped from the membrane by treatment with the chaotropic agent KI, confirming it to be part of the soluble complex of the V-ATPase. In immunoblots, the 14-kDa-specific antibodies showed no cross-reaction with several xenic V-ATPases.

Characterization of proton transport in bone-derived membrane vesicles

ATP-dependent proton transport in membrane vesicles prepared from the medullary bone of egg-laying hens, a source rich in osteoclasts, was characterized. Proton transport was abolished by bafilomycin A 1 (10 nM) and N-ethylmaleimide (50 μM), but not by oligomycin (15 μg/ml), vanadate (100 μM) or SCH 28080 (100 μM), thereby differentiating this H +-ATPase from the F 1F 0- and phosphorylated-type of ATPases. Preincubation of the membrane vesicles at 0°C for 1 h in the presence of KCl (0.3 M) and Mg-ATP (5 mM) resulted in almost complete loss of H +-transport activity (cold-inactivation). Preventing the formation of a membrane potential by voltage clamp ( K in + = K out + + valinomycin) increased both the rate of H +-transport and the equilibrium ΔpH, suggesting an electrogenic proton transport mechanism. Thus, the H +-ATPase in this bone-derived membrane vesicle preparation shows the characteristics of a vacuolar H +-ATPase in its inhibitor- and cold-sensitivity and its electrogenic mechanism. The anion sensitivity of the H +-ATPase was investigated by varying the intra- and/or extra-vesicular salt composition. The H +-ATPase had no absolute requirement for any specific anion, but membrane permeable anions were found to stimulate proton transport activity, presumably by acting as charge compensators for the electrogenic hydrogen ion transport. However, some anions, such as sulfate, acetate and nitrate were directly inhibitory to the ATPase. The results are in agreement with the recently proposed mechanism of osteoclast acidification: a vacuolar H +-ATPase working in parallel with a Cl −-channel resulting in electroneutral HCl secretion.

Characterization of ATP-dependent proton transport in medullary bone-derived microsomes

Proton transport in microsomal vesicles derived from medullary bone of laying hens was observed to be inhibited in a dose-dependent manner with fusidic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diatzole (NBD-Cl), duramycin and dicyclohexylcarbodiimide (DCCD). The IC50 values were 570 microM, 4.5 microM, 10 micrograms/ml and 32 microM for fusidic acid, NBD-Cl, duramycin and DCCD, respectively. 14C-DCCD labeled a single protein band of 15-17 kDa from bone-derived microsomes in SDS-electrophoresis. A protein of this size is a proton-conducting subunit of the vacuolar ATPases. Further, the proton transport was found to be electrogenic, thus it generates the membrane potential across the vesicle membrane. The generation of membrane potential was inhibited using 100 nM bafilomycin A1, which in low concentrations is a specific inhibitor of vacuolar ATPases. The presence of Cl- was essential for maximal proton transport activity. These results confirm the electrogenicity and extend the characterization of the osteoclastic H(+)-ATPase.

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.

Steady-state kinetics of the plasma membrane H+-ATPase from red beet (Beta vulgaris): its inhibition by vanadate and inorganic phosphate

The intial velocity vs ATP concentration curves obtained with the plasma membrane H+-ATPase from red beet (Beta vulgaris L.) did not follow classical Michaelis-Menten kinetics. A rate equation containing second-order terms in ATP concentration in both the numerator and the denominator was used to obtain a significantly better fit to the data. The observed deviations from Michaelis-Menten kinetics were more pronounced in the presence of potassium ions. The inhibition caused by inorganic phosphate was partial. i.e. the ATPase activity extrapolated at an infinite phosphate concentration was not zero. In contrast, the inhibition produced by orthovanadate was nearly total. The inhibitions caused by both phosphate and vanadate were uncompetitive with respect to ATP and enhanced by potassium ions and high concentrations of dimethyl sulfoxide. a solvent used to lower the water activity of the reaction medium. The ATP-dependent proton transport was stimulated by potassium ions and was inhibited by phosphate only at high ATP concentrations. A kinetic mechanism, in which the H+-ATPase can adopt two conformations during its catalytic cycle and can form a ternary enzyme-ATP-phosphate complex able to hydrolyze bound ATP. is proposed to explain those results.

Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro.

Bisphosphonates are useful in treatment of disorders with increased osteoclastic activity, but the mechanism by which bisphosphonates act is unknown. We used cultures of chicken osteoclasts to address this issue, and found that 1-hydroxyethylidenediphosphonic acid (EHDP), dichloromethylidenediphosphonic acid (Cl2MDP), or 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid (APD) all cause direct dose-dependent suppression of osteoclastic activity. Effects are mediated by bone-bound drugs, with 50% reduction of bone degradation occurring at 500 nM to 5 microM of the different agents. Osteoclastic bone-binding capacity decreased by 30-40% after 72 h of bisphosphonate treatment, despite maintenance of cell viability. Significant inhibition of bone resorption in each case is seen only after 24-72 h of treatment. Osteoclast activity depends on ATP-dependent proton transport. Using acridine orange as an indicator, we found that EHDP reduces proton accumulation by osteoclasts. However, inside-out plasma membrane vesicles from osteoclasts transport H+ normally in response to ATP in high concentrations of EHDP, Cl2MDP, or APD. This suggests that the bisphosphonates act as metabolic inhibitors. Bisphosphonates reduce osteoclastic protein synthesis, supporting this hypothesis. Furthermore, [3H]leucine incorporation by the fibroblast, which does not resorb bone, is also diminished by EHDP, Cl2MDP and APD except when co-cultured with bisphosphonate-binding bone particles. Thus, the resorption-antagonizing capacities of EHDP, Cl2MDP and APD reflect metabolic inhibition, with selectivity for the osteoclast resulting from high affinity binding to bone mineral.

Acidification and Ion Permeabilities of Highly Purified Rat Liver Endosomes

While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.

Inhibition of proton transport activity of microsomal membrane vesicles of barley roots by aluminium

Abstract The extrusion of protons from the roots of barley (Hordeum vulgare L.) seedlings in the presence of KCl decreased markedly after treatment of the samples with 1 mm AlCl3 at pH 5.5. The membrane vesicles with ATP-dependent proton transport activity were prepared from barley roots. The proton transport activity measured by the quenching of the fluorescence of quinacrine was inhibited by approximately 50% in the presence of 100 μm AlCl3 at pH 6.5 but the inhibition was only 10% at pH 7.5. The values of V max and K m for ATP related to the proton transport activity of the vesicles were affected markedly by AlCl3. The inhibition of the proton transport activity of the vesicles due to Al was effectively restored by citric acid. The results suggest that one of the mechanism of Al toxicity in plant roots is the inhibition of the proton transport activity associated with the cell membrane which in turn leads to the inhibition of proton extrusion from the roots.

Separation and Immunological Characterization of Membrane Fractions from Barley Roots

Tonoplast and plasma membranes (PM) were isolated from barley roots (Hordeum vulgare L. cv California Mariout 72) using sucrose step gradients. The isolation procedure yielded sufficient quantities of PM and tonoplast vesicles that were sealed and of the right orientation to measure ATP-dependent proton transport in vitro. The proteins of the endoplasmic reticulum, tonoplast-plus-Golgi membrane (TG) and PM fractions were separated on sodium dodecyl sulfate gels, and immunoblots were used to test for cross-contamination between the fractions. Proteins that cross-reacted with antibodies to the PM ATPase from corn roots and Neurospora were greatly enriched in the PM fraction, as were proteins that cross-reacted with monoclonal antibodies to an arabinogalactan protein from the PM of tobacco cells. Proteins that cross-reacted with antibodies to the 58- and 72-kilodalton subunits of the tonoplast ATPase of red beet storage tissue were greatly enriched in the TG fraction. The results with immunoblots and enzyme assays indicated that there was little cross-contamination between the tonoplast and PM vesicles. The molecular weights and isoelectric points of the PM ATPase and the tonoplast ATPase subunits were also determined using immunoblots of two-dimensional gels of the PM and TG proteins.

Proton-translocating ATPase from bovine kidney medulla: partial purification and reconstitution.

The proton-translocating ATPase that is responsible both for urinary and vacuolar acidification was partially purified from bovine kidney medulla microsomes. ATPase activity was purified to a maximum specific activity of 1.7 mumol.min-1.mg prot-1 and was inhibited completely by N-ethylmaleimide. The relative molecular weight (Mr) of the intact protein estimated by high-pressure size-exclusion liquid chromatography was 586,000. Nondenaturing gels of the isolated enzyme revealed two protein bands at MrS of 551,000 and 523,000. Sodium dodecyl sulfate-gel electrophoresis of the isolated H+-ATPase revealed component subunits at MrS of 70,000, 56,000, 45,000, 42,000, 38,000, 31,000, 15,000, 14,000, and 12,000. The properties of the isolated H+-ATPase and of microsomal ATP-dependent proton transport correlated closely. The isolated H+-ATPase was reconstituted into phospholipid liposomes and demonstrated N-ethylmaleimide-inhibitable ATP-dependent potential generation, consistent with electrogenic proton transport. In overall structure, the enzyme appears to be a new type of H+-ATPase with several features of the F0F1 class of ion-translocating ATPases but is immunologically and structurally different from the mitochondrial F1-ATPase.

Vanadate inhibition of ATP-dependent H + transport in membrane vesicles from turtle bladder epithelial cells

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO(3) (-) and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H(+)-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO(3) when assayed at 24 degrees C or above and was tentatively identified as the tonoplast H(+)-ATPase. A smaller peak of H(+)-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO(3) and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H(+)-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO(3) inhibited ATP hydrolysis by the tonoplast ATPase at 12 degrees C and the initial rate of proton transport was stimulated by 50 millimolar KNO(3). The time course for fluorescence quench indicated that addition of ATP in the presence of KNO(3) caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO(3) was explained by the temperature-dependent delay of the inhibition by KNO(3). The plasma membrane H(+)-ATPase maintained a pH gradient in the presence of KNO(3) for up to 30 minutes at 24 degrees C.

Identification of a vanadate-sensitive potassium-dependent proton pump from rabbit colon.

Membrane vesicles prepared from rabbit distal colon were used to study colonic transport mechanisms. Differential and sucrose-Ficoll density gradient centrifugation of the mucosal homogenate yielded fractions which supported ATP-dependent proton transport, as measured with the fluorescent weak base acridine orange. Quenching of acridine orange absorbance in light microsomes and microsome-derived density gradient fractions was MgATP-dependent and was reversed with nigericin; these characteristics suggest the presence of one or more ATP-driven proton pumps. Proton transport in the microsomal fraction was inhibited by N-ethylmaleimide more than by orthovanadate, and was dependent on extravesicular chloride. Vesicles in a microsome-derived gradient fraction were inhibited by orthovanadate more than by N-ethylmaleimide. N-ethylmaleimide pretreatment of this gradient fraction uncovered a vesicle population with characteristics similar to the gastric H+,K+ATPase: proton transport was abolished by orthovanadate and the experimental anti-ulcer drug SCH 28080, was enhanced by potassium, and was not affected by chloride. ATP-generated proton gradients in this fraction were not dissipated by the proton ionophore 3,3',4',5-tetrachlorosalicylanilide. We conclude that two ATP-driven proton pumps are present in mucosa from distal rabbit colon; one with characteristics of N-ethylmaleimide-sensitive organelle associated proton pumps, and one similar to the gastric proton-potassium exchanger.

Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron.

This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.

Processing and secretion in the neurohypophysis. Stability of isolated secretory vesicles and role of internal pH

A possible role of low pH in secretory vesicles for processing and secretion in the neurohypophysis was investigated. Subcellular fractionation of guinea-pig neural lobes revealed that a proton present in the membranes from this tissue could not be ascribed to secretory vesicles. However, a proton pump was found in coated microvesicles. Secretory vesicles isolated from rats and guinea pigs were stable under conditions known to lyse secretory vesicles from the adrenal medulla owing to the generation of a proton gradient. These results suggest that the internal pH of secretory vesicles from the neurohypophysis is closer to neutral than is the pH in chromaffin secretory vesicles. Processing of a neurophysin-glycopeptide intermediate from the biosynthesis of vasopressin in intact secretory vesicles incubated in vitro was activated by the addition of NH4Cl, known to increase the intravesicular pH. This activation of neurohormone processing was also apparent in isolated nerve endings incubated in the presence of NH4Cl, suggesting that NH4Cl can also be used to increase the intravesicular pH in intact nerve endings. However, NH4Cl did not affect the secretion of neurohormones, indicating that a low intravesicular pH is not important for exocytosis in the neurohypophysis. Our results indicate that a low pH generated during processing by mechanisms other than ATP-dependent proton transport may inhibit the processing enzymes, thereby preventing extensive breakdown of neurohormone precursors.