NEM-sensitive ATPase Activity Research Articles

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Disassembly of the yeast V-ATPase into cytosolic V(1) and membrane V(0) sectors inactivates MgATPase activity of the V(1)-ATPase. This inactivation requires the V(1) H subunit (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767), but its mechanism is not fully understood. The H subunit has two domains. Interactions of each domain with V(1) and V(0) subunits were identified by two-hybrid assay. The B subunit of the V(1) catalytic headgroup interacted with the H subunit N-terminal domain (H-NT), and the C-terminal domain (H-CT) interacted with V(1) subunits B, E (peripheral stalk), and D (central stalk), and the cytosolic N-terminal domain of V(0) subunit Vph1p. V(1)-ATPase complexes from yeast expressing H-NT are partially inhibited, exhibiting 26% the MgATPase activity of complexes with no H subunit. The H-CT domain does not copurify with V(1) when expressed in yeast, but the bacterially expressed and purified H-CT domain inhibits MgATPase activity in V(1) lacking H almost as well as the full-length H subunit. Binding of full-length H subunit to V(1) was more stable than binding of either H-NT or H-CT, suggesting that both domains contribute to binding and inhibition. Intact H and H-CT can bind to the expressed N-terminal domain of Vph1p, but this fragment of Vph1p does not bind to V(1) complexes containing subunit H. We propose that upon disassembly, the H subunit undergoes a conformational change that inhibits V(1)-ATPase activity and precludes V(0) interactions.

Gill ATPase activities of silver perch, Bidyanus bidyanus (Mitchell), and golden perch, Macquaria ambigua (Richardson): Effects of environmental salt and ammonia

The Australian freshwater fish, silver and golden perch, are increasingly being used for aquaculture. Addition of salt to water is commonly used in commercial aquaculture to reduce stress attributed to high ammonia concentrations. The activities in gill homogenates of ouabain-sensitive Na +/K +-ATPase and NEM-sensitive ATPases (as a measure of H +-ATPases) of silver and golden perch were measured after maintaining the fish in water containing different salt and ammonia concentrations. Six treatments were applied in a 2 × 3 factorial design: two salt treatments, low salt (LS) of 2.5 g l − 1 and high salt (HS) 5 g l − 1 , and three ammonia treatments, no added ammonia (NA), low ammonia (LA), 3 mg total ammonia nitrogen (TAN) l − 1 and high ammonia (HA), 5 mg TAN l − 1 . In both species, activity of Na +/K +-ATPase was lowest in fish kept in the LSNA treatment (7.4 ± 0.4 μmol Pi mg protein − 1 h − 1 for silver perch and 3.1 ± 0.6 for golden perch) and highest in the HSHA treatment (15.2 ± 1.0 μmol Pi mg − 1 protein h − 1 for silver and 8.4 ± 1.2 for golden perch). In both species there was a significant increase ( P < 0.001) in Na +/K +-ATPase activity with increase in salt concentration and with an increase in ammonia concentrations. A significant interaction ( P < 0.036) between salt and ammonia on Na +/K +-ATPase activity was observed in silver but not in golden perch. In contrast, the lowest activity for NEM-sensitive ATPase was observed in the HSNA treatment (1.0 ± 0.2 μmol Pi mg − 1 protein h − 1 for silver and 1.5 ± 0.4 for golden perch) and highest in LSHA treatment (2.9 ± 0.4 μmol Pi mg − 1 protein h − 1 for silver and 3.6 ± 1.2 for golden perch). In both species there was a significant decrease in NEM-sensitive ATPase activity with increase in salt concentration and an increase in activity with increase in ammonia ( P < 0.003). In silver perch, a significant interaction between the treatments was observed ( P < 0.02). The results suggest that in these species of freshwater fish the Na +/K +-ATPase has a role in salt and ammonia homeostasis and that the NEM-sensitive ATPases are more active in fish kept in water with a lower salt content. It is possible that the increase in ammonia resistance when salt is added to the environmental water in commercial aquaculture systems may be due to the effects of salt on gill Na +/K +-ATPase activity rather than the NEM-sensitive ATPases.

H+ -ATPase Activity in Crude Homogenates of Fish Gill Tissue: Inhibitor Sensitivity and Environmental and Hormonal Regulation

ABSTRACT N-ethymaleimide-sensitive ATPase activity was measured in crude homogenates of gill tissue from rainbow trout using a coupled-enzyme ATPase assay in the presence of EGTA, ouabain and azide. This NEM-sensitive ATPase activity, determined to be about 1.5 μmolmg−1 protein h−1 at 15°C for freshwater trout, is also inhibited by other H+-ATPase blockers such as DCCD, DES, PCMBS and bafilomycin. It is concluded, therefore, that the NEM-sensitive ATPase activity was generated by a proton-translocating ATPase. Since this NEM-sensitive ATPase was also sensitive to the plasma membrane ATPase inhibitor vanadate, we conclude that the H+-ATPase in fish gill is of the plasma membrane type. The major role of the H+-ATPase in the gill epithelium is to facilitate Na+ uptake from fresh water. Sodium concentration in the external medium was the primary regulator of the H+-ATPase in fish gills, with low sodium levels being associated with high H+-ATPase activity. High external calcium concentration had a marked stimulatory effect on H+-ATPase activity in fish gills when the sodium level was low. Environmental hypercapnia induced a 70% increase in the H+-ATPase activity in fish gills. H+-ATPase activity was also elevated in freshwater fish after chronic cortisol infusion.

Major ATPases on clofibrate-induced rat liver peroxisomes are not associated with 70 kDa peroxisomal membrane protein (PMP70).

We previously reported that novel Mg(2+)-ATPases were induced in rat liver peroxisomes by clofibrate administration and that these activities consisted of at least two types of enzymes, N-ethylmaleimide (NEM)-sensitive and -resistant. Here we present evidence that neither of these major peroxisomal ATPases is associated with the 70-kDa peroxisomal membrane protein (PMP70), because: (i) proteinase K treatment of peroxisomes resulted in inactivation of only NEM-sensitive ATPase, whereas disappeared PMP70 completely; (ii) NEM-sensitive ATPase activity was barely immunoprecipitated with anti-PMP70 IgG; (iii) the solubilized ATPases behaved differently from PMP70 on native PAGE; and finally (iv), the major peroxisomal ATPases were separated from PMP70 on gel filtration chromatography.

Evidence for an ATP-driven H +-pump in the plasma membrane of the bovine corneal epithelium

In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2·7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-β- d-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H +-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H +-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H +-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H +-pump activity was: ATP ⪢ GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg 2+ with Mn 2+ did not change the acidification rate but Co 2+ only partly activated whereas Ca 2+ and Zn 2+ were ineffective as activators. The H +-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 μ m NEM or NBD-Cl and 92% inhibited by 20 μ m DCCD. The pH dependency of the NEM-sensitive ATPase activity showed a single optimum pH at 7·5. It is concluded that a major portion of the H +-pump activity in this membrane preparation is derived from the plasma membrane and that the properties of this plasma membrane H +-pump are similar to those of the vacuolar (V-type) H +-pumps described in various intracellular organelles and in the plasma membrane of other cell types.

Effect of metabolic acidosis and alkalosis on NEM-sensitive ATPase in rat nephron segments.

An N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) displaying the kinetic and pharmacological properties of an electrogenic proton pump has been described in the different segments of rat nephron, where it mediates part of the active tubular proton secretion. This study was therefore designed to evaluate whether changes in urinary acidification observed during metabolic acidosis or alkalosis were associated with alterations of the activity of tubular NEM-sensitive ATPase, and if so, to localize the nephron segments responsible for these changes. Within 1 wk after the onset of ammonium chloride treatment, rats developed a metabolic acidosis, and NEM-sensitive ATPase activity was markedly increased in the medullary thick ascending limb of Henle's loop and outer medullary collecting tubule, and slightly increased in the cortical collecting tubule. Conversely, treatment with sodium bicarbonate induced a metabolic alkalosis that was accompanied by decreased NEM-sensitive ATPase activity in medullary thick ascending limb and outer medullary collecting tubule. NEM-sensitive ATPase activity was not altered in any other nephron segment tested in alkalotic and acidotic rats, i.e., the proximal tubule and the cortical thick ascending limb of Henle's loop. Changes qualitatively similar were observed as soon as 3 h after the onset of NaHCO3 or NH4Cl-loading. In the medullary collecting tubule, alterations of NEM-sensitive ATPase activity are in part due to hyperaldosteronism observed in both acidotic and alkalotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease in N-ethylmaleimide-sensitive ATPase activity in collecting duct by metabolic alkalosis

Changes in systemic acid-base balance are known to influence acidification in the collecting duct. The H+ secretion in the collecting duct has been shown to be an electrogenic process and it has been suggested that an H-ATPase sensitive to inhibition by N-ethylmaleimide (NEM) is responsible for H+ secretion. This study was designed to determine the effect of metabolic alkalosis on NEM-sensitive ATPase activity in the microdissected segments of the distal nephron. Metabolic alkalosis was produced by giving NaHCO3 to normal rats for 7 days. The plasma total CO2 concentration in the experimental group was 31.5 +/- 1.8 mM compared with 23.4 +/- 1.0 mM in the control group. NEM-sensitive ATPase activity was significantly lower in the cortical collecting duct and in the outer and inner medullary collecting ducts of alkali-loaded rats than those of control rats. There was no significant difference in the enzyme activity between the two groups of animals in the other nephron segments examined. Our results suggest that NEM-sensitive H-APTase activity in all three segments of the collecting duct is modulated by the acid-base status of the animal.

NEM-sensitive ATPase activity in rat nephron: effect of metabolic acidosis and alkalosis

The present study was designed to quantitate the amount and to map the localization of N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) activity in microdissected segments of the rat nephron. After complete nephron mapping the effect of chronic metabolic acidosis and alkalosis on enzyme activity was determined. In control animals the highest enzyme activity was found in the early proximal convoluted tubule of juxtamedullary nephrons; superficial early proximal tubule as well as medullary and cortical thick ascending limbs and collecting ducts also contained substantial activity. Enzyme activity in the papillary collecting duct before entry into the ducts of Bellini was 329 +/- 93 pmol.mm-1.h-1 (n = 8); after entry, however, enzyme activity was approximately one-fourth that value (60 +/- 9 pmol.mm-1.h-1, n = 8, P less than 0.01). No NEM-sensitive ATPase activity was found in the thin limbs of the loop of Henle. Enzyme activity increased in both the medullary and cortical thick ascending limbs as well as in the cortical collecting tubule in response to NH4Cl-induced chronic metabolic acidosis; in the cortical collecting duct, metabolic acidosis increased maximum activity (Vmax) but did not change Michaelis-Menten constant (Km). In the proximal convoluted tubule, enzyme activity decreased with metabolic acidosis. Bicarbonate loading had no effect on enzyme activity except in the most distal portion of the collecting duct where it was stimulated. These results show that NEM-sensitive ATPase activity exists throughout much of the rat nephron. These data suggest that both the cortical collecting tubule and thick ascending limb are regulatory sites of distal urinary acidification during acid loading.

Effects of Low-Potassium Diet on N-Ethylmaleimide-Sensitive ATPase in the Distal Nephron Segments

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.

Enzyme activity in obstructive uropathy: Basis for salt wastage and the acidification defect

Unilateral ureteral obstruction results in marked changes in renal function throughout the nephron, including impaired acid and potassium secretion and salt wastage. The nephron site believed responsible for the acidification defect is the collecting duct. It has been presumed, although not demonstrated, that the cellular mechanism for the acidification defect is both a decrease in transepithelial voltage and a decrease in activity of the proton pump located at the luminal membrane. The mechanism for the abnormalities in sodium handling are thought due to alterations in Na-K ATPase activity. Our laboratory has recently mapped the profile of the N-ethylmaleimide (NEM)-sensitive ATPase and Na-K ATPase in microdissected rat nephron, documenting their presence throughout much of the nephron. In animals with acute unilateral ureteral obstruction for 18 to 24 hours, we measured NEM-sensitive ATPase and Na-K ATPase activities in several nephron sites. In all nephron segments Na-K ATPase activity was markedly decreased. In the medullary collecting duct, NEM-sensitive ATPase activity was also markedly reduced in animals with acute ureteral obstruction; in the cortical collecting duct, activity fell significantly, but to a lesser degree than was observed in the medullary collecting duct. NEM-sensitive ATPase activity was unchanged from control in the proximal convoluted tubule and in the medullary thick ascending limb; in the cortical thick ascending limb enzyme activity increased. These results demonstrate a change in both Na-K ATPase and NEM-sensitive ATPase activities as a direct consequence of a defect known to result in salt wastage and an acidification defect in humans and animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term effect of aldosterone on NEM-sensitive ATPase in rat collecting tubule

Because previous studies indicated that in the collecting tubule, N-ethylmaleimide (NEM)-sensitive ATPase, the biochemical equivalent of the proton pump, is controlled by mineralocorticoids in the long term, the present study was designed to investigate whether such control also exists in the short term. Therefore we investigated the in vivo and in vitro effects of aldosterone on the enzyme activity in cortical and outer medullary collecting tubules (CCT and MCT, respectively) from adrenalectomized rats. Administration of aldosterone (10 micrograms/kg body wt) markedly stimulated NEM-sensitive ATPase activity in the CCT and MCT within 3 h. Similarly, incubating CCT or MCT for 3 h in the presence of 10(-8) M aldosterone enhanced NEM-sensitive ATPase activity up to values similar to those previously measured in the corresponding nephron segments of normal rats. In vitro stimulation of NEM-sensitive ATPase was dose dependent in regard to aldosterone (apparent affinity constant approximately 10(-9) M), appeared after a 30-min lag period, and reached its maximum after 2-2.5 h. Finally, actinomycin D and cycloheximide totally abolished the in vitro action of aldosterone, demonstrating the involvement of protein synthesis in this process.

Effects of aldosterone on NEM-sensitive ATPase in rabbit nephron segments

Aldosterone (aldo) treatment of animals stimulates the rate of H+ secretion in the collecting duct, a process which may involve an H+-ATPase sensitive to inhibition by NEM (N-ethylmaleimide). Therefore, we determined NEM-sensitive ATPase activity in distal nephron segments from three groups of adrenalectomized (adx) rabbits maintained on different doses of aldo (in an osmotic minipump) for seven days. Group 1 was given 1.5 micrograms aldo/100 g body wt/day, whereas groups 2 and 3 were maintained on 5 micrograms and 50 micrograms of aldo/100 g body wt/day, respectively. Aldo concentrations in the plasma of groups 1, 2 and 3 were 10.4 +/- 0.8, 70 +/- 7 and 408 +/- 133 ng/dl, respectively. There was a significant increase in NEM-sensitive ATPase activity in connecting tubule (CNT) and cortical, outer and inner medullary duct segments (CCD, OMCD and IMCD) but not in cortical thick ascending limb (CTAL) and distal convoluted tubule (DCT) in group 2 as compared to group 1. A further increase in plasma concentration of aldo (group 3) did not produce any more increase in NEM-sensitive ATPase activity in the CNT, CCD, OMCD and IMCD, but did increase the enzyme activity in the DCT. These results are consistent with the hypothesis that aldo increases H+ secretion in the connecting tubule and collecting duct segments by increasing the activity of NEM-sensitive H+-ATPase activity in these segments.

Effect of adrenalectomy on NEM-sensitive ATPase along rat nephron and on urinary acidification.

An N-ethyl-maleimide (NEM)-sensitive ATPase that displays the properties of an electrogenic proton pump has been described in the different segments of the rat nephron where it mediates part of the active tubular proton secretion. Because corticosteroids are known to control kidney acidification, we evaluated whether or not NEM-sensitive ATPase is a target of corticosteroids in some nephron segments. For this purpose we measured NEM-sensitive ATPase activity in the different segments of nephron microdissected from normal and adrenalectomized rats. Results indicate that within 1 wk after adrenalectomy NEM-sensitive ATPase activity was markedly decreased in both cortical and outer medullary portions of the collecting tubule (cortex, from 398 +/- 12 (+/-SE) to 145 +/- 20; outer medulla, from 293 +/- 21 to 112 +/- 14 pmol X mm-1 X h-1); however, it was not altered in any other segment of the nephron. These results demonstrate that kidney NEM-sensitive ATPase is under the control of corticosteroids and suggest that mineralocorticoids rather than glucocorticoids are involved in this regulation that specifically occurs in mineralocorticoid-sensitive nephron segments. This paper also describes a new computerized method for the automatic determination of the length of single nephron segments.

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.

Characterization of native and reconstituted hydrogen ion-pumping adenosine triphosphatase of chromaffin granules

The ATP-dependent H+ pump from adrenal chromaffin granules is, like the platelet-dense granule H+ pump, essentially insensitive to the mitochondrial ATPase inhibitors sodium azide, efrapeptin, and oligomycin and also insensitive to vanadate and ouabain, agents that inhibit the Na+,K+-ATPase. The chromaffin granule H+ pump is, however, sensitive to low concentrations of NEM (N-ethylmaleimide) and Nbd-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). These transport ATPases may thus belong to a new class of ATP-dependent ion pumps distinct from F1F0-and phosphoenzyme-type ATPases. Comparisons of ATP hydrolysis with ATP-dependent serotonin transport suggest that approximately 80% of the ATPase activity in purified chromaffin granule membranes is coupled to H+ pumping. Most of the remaining ATPase activity is due to contaminating mitochondrial ATPase and Na+,K+-ATPase. When extracted with cholate and octyl glucoside, the H+ pump is solubilized in a monodisperse form that retains NEM-sensitive ATPase activity. When reconstituted into proteoliposomes with crude brain phospholipid, the extracted enzyme recovers ATP-dependent H+ pumping, which shows the same inhibitor sensitivity and nucleotide dependence as the native pump. These data demonstrate that the predominant ATP hydrolase of chromaffin granule membrane is also responsible for ATP-driven amine transport and granule acidification in both native and reconstituted membranes.

Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis.

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10-35 pmol ADP X min-1 X mm-1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duct segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 +/- 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60-100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.

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.