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.     

Evidence for the presence of H+-ATPase in the membrane of brain synaptic vesicles in the rat

Mg-ATPase of rat brain synaptic vesicles (SV) is considerably (by 85%) inhibited by dicyclohexyl carbodiimide (200 microM), a blocker of proton pumps, whereas orthovanadate (100 microM) does not produce any influence on the enzyme. Oligomycin (5 micrograms/ml) does not alter Mg-ATPase activity of the SV, whereas N-ethylmaleimide (300 microM) reduces it to a moderate degree, namely by 35%. This indicates that Mg-ATPase of the SV differs from mitochondrial ATPase. The protonophore p-trichloromethoxycarbonyl cyanide phenylhydrazone (20 microM) and bicarbonate anions (20 mM) stimulate slightly (by 12 to 25%) Mg-ATPase of the SV. Bicarbonate (20 mM) raises 1.8-2.1-fold Mg-ATPase activity of the mitochondria isolated from rat brain. It is assumed that the membrane of brain SV contains proton ATPase (H+-ATPase) differing from mitochondrial H+-ATPase in some of the properties.     

Proton translocation coefficient for H+-ATPase of rat liver mitochondria

Some features of H+-ATPase function in intact mitochondria of rat liver were studied. Simultaneously the activities of ATPase and proton translocase were measured, using a previously described technique. The proton translocation coefficient of H+-ATPase has been found to be equal to 3.6. The protonophore 3.5-di-tert-butyl-4-hydroxybenzylidenemalononitrile diminishes the proton translocation coefficient. It was concluded that when considering the mechanism of proton translocation by H+-ATPase, it is necessary to assume the possibility of transport of 3 or 4 protons per every hydrolyzed molecule of ATP allowing a changeable efficiency of the process. The decrease of the translocase coefficient in the presence of the protonophore appears to result from the ability of this uncoupler to return the transferred protons to the mitochondrial matrix.     

Lability of coupling between proton translocase and ATPase of mitochondrial H+-ATPase complex]

The interrelationship between the ATPase and H+-translocase functions of mitochondrial H+-ATPase was studied. The efficiency of the functioning was estimated by the value of coupling coefficient (Kc), which is represented by a ratio of proton translocation rate versus ATP coupling hydrolysis rate. It was shown that under conditions of increased concentrations of ATP and low concentrations of oligomycin the value of Kc is decreased. The increase in the concentration of valinomycin results in an increase of Kc. It was also found that the H+-ATPase activity shows a considerable increase during incubation of mitochondria, reaching its maximum with respect to both functions 1--2 min after addition of ATP. The data obtained are indicative of a lack of tight coupling between the H+-translocase and ATPase functions of mitochondrial H+-ATPase. The mechanism of action of H+-ATPase is discussed.     

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

The effect of the natural protein inhibitor on H+-ATPase hepatoma 22a mitochondria

The uncoupler-induced inactivation of H+-ATPase in hepatoma 22a and mouse liver mitochondria has been studied. The dependence of this process on delta microH, and pH and ATP was established. The inactivated ATPase could be reactivated at alkaline pH values in the absence of ATP. These data indicate that the inactivation is apparently caused by the natural protein inhibitor. ATP- and pH-dependent decrease of ATPase activity is also observed after Lubrol-WX disruption of mitochondria. It can be proposed that practically all ATPase molecules in hepatoma mitochondria are in a catalytically active complex with the protein inhibitor. At low delta microH this complex is inactivated via reversible pH-dependent and irreversible ATP-dependent rearrangements. The pH-dependent rearrangement of the isolated protein inhibitor from hepatoma mitochondria is also observed.     

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.     

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.     

Amplification of the Streptococcus faecalis proton-translocating ATPase by a decrease in cytoplasmic pH

When Streptococcus faecalis was grown in the presence of protonophores , an ATPase activity of the membrane was increased at a pH below 8.0 but not at a pH above 8.0. Characteristics of this increased ATPase were identical to those of a proton-translocating ATPase (H+-ATPase) located on the membrane of normal cells. The cytoplasmic pH was regulated at 7.6 to 7.8 but was not regulated in the presence of protonophores . The increase in the H+-ATPase was observed when the cytoplasmic pH was lowered to less than 7.6 by the addition of protonophores and was not related to the dissipation of the proton motive force. Thus, we suggest that the H+-ATPase of the membrane is amplified when the cytoplasmic pH is lowered below the pH at which it is regulated under normal conditions.     

Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea. I. Characterization of proton pumping and ATPase activity

Sealed vesicles were isolated from a plant pathogenic fungus Phytophthora megasperma f. sp. glycinea using a modification of a method previously developed for plant plasma membrane vesicle isolation. Vanadate-sensitive, proton pumping microsomal membrane vesicles were resolved on a linear sucrose density gradient and found to comigrate with a vanadate-sensitive ATPase. Both the proton pumping and ATPase activity of these vesicles had a pH optimum of 6.5 and demonstrated similar properties with respect to substrate specificity and inhibitor sensitivity. These properties were in agreement with previously published data on the Phytophthora plasma membrane ATPase. In contrast with previous reports there was no K+ stimulation of the plasma membrane ATPase and the Km for Mg:ATP (1:1 concentration ratio) was higher (2.5 mM). A comparison of anion (potassium salts) effects upon delta pH and delta psi formation in sealed Phytophthora plasma membrane vesicles revealed a correspondence between the relative ability of anions to stimulate proton transport and to reduce delta psi. The relative order for this effect was KCl greater than KBr much greater than KMes, KNO3, KClO3, K2SO4. This study presents a method for the isolation of sealed vesicles from Phytophthora hyphae. It also provides basic information on the plasma membrane H+-ATPase and its associated proton pumping activity.     

Inhibitor peptide of mitochondrial proton adenosine triphosphatase. Neutralization of its inhibitory action by calmodulin

In the presence of ATP and Mg2+, the homogeneous ATPase peptide inhibitor of rat liver mitochondria markedly inhibits the proton ATPase from this source (Cintrón N. M., and Pedersen, P. L. (1979) J. Biol. Chem. 254, 3439-3443). Under these conditions, calmodulin prevents the inhibitor peptide from inhibiting the liver H+-ATPase. About 1.5 mol of calmodulin/mol of inhibitor is necessary to effect a half-maximal response (apparent Km = 0.5 microM calmodulin). The capacity of calmodulin to neutralize the action of the ATPase inhibitor peptide appears highly specific. This effect is not produced by insulin, trypsin inhibitor, lysozyme, ribonuclease, myoglobin, cytochrome c, ovalbumin, or bovine albumin. Only polyglutamate was found to mimic the action of calmodulin. However, when added together with calmodulin, polyglutamate failed to elicit an additive effect indicating that its site of interaction on the ATPase inhibitor peptide differs from that of calmodulin. Calcium is not essential in the assay medium for calmodulin to neutralize the action of the ATPase inhibitor peptide. The neutralization effect produced by calmodulin is also source-independent, with preparations of calmodulin from bovine brain and rat testes being equally competent. Calmodulin has no direct effect on the ATPase activity of the proton ATPase, nor does it affect the capacity of the enzyme to participate in either ATP synthesis or the ATP-dependent transhydrogenase reaction. Moreover, calmodulin fails to reverse inhibition of the H+-ATPase to which ATPase inhibitor peptide is already bound. Overall, these results indicate that calmodulin interacts in a direct and highly specific manner with the "free" ATPase peptide inhibitor of rat liver mitochondria.     

Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae

The thermosensitive G1-arrested cdc35-10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature. After 1 h incubation at this temperature, the plasma membrane H+-ATPase activity of cdc35-10 was reduced to 50%, whereas that in mitochondria doubled. Similar data were obtained with cdc25, another thermosensitive G1-arrested mutant modified in the cAMP pathway. In contrast, the ATPase activities of the G1-arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature. In the double mutants cdc35-10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35-10 and cdc25. These data indicate that cAMP acts as a positive effector on the H+-ATPase activity of plasma membranes and as a negative effector on that of mitochondria.     

Identification of a Ca2+-ATPase in brown adipose tissue mitochondria: regulation of thermogenesis by ATP and Ca2+

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca(2+) concentration from 0.05 up to 0.70 mum. It is not known how the rise of Ca(2+) concentration activates BAT thermogenesis. In this report we compared the effects of Ca(2+) in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase bound to the inner membrane of BAT mitochondria. A Ca(2+)-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca(2+) enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca(2+) concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 mum. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca(2+) did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca(2+)-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca(2+) concentrations similar to those noted in the cell during adrenergic stimulation.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

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.     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

Changes in the membrane environment of the (K+ + H+)-ATPase following stimulation of the gastric oxyntic cell

Biochemical evidence is presented for changes in the membrane environment of the (K+ + H+)-dependent ATPase enzyme of the oxyntic cell following in vivo gastric stimulation of young New Zealand rabbits. The changes are inferred from the marked differences in the sedimentation properties of the (K+ + H+)-ATPase when obtained from homogenates of either stimulated or nonstimulated (resting) fundic gastric epithelium. Stimulation resulted in a redistribution of K+-ATPase activity that was reduced to less than half in the microsomal pellet and concomitantly increased in the membrane fractions normally associated with nuclei and mitochondria. Density gradient fractionation of the mitochondrial pellet yield a preparation rich in (K+ + H+)-ATPase. Our studies indicated that the membranes in this preparation are far larger and apparently denser than the microsomal vesicles associated with the nonstimulated state of the cell. The specific nature of the relationship between stimulation and the observed changes is suggested by the lack of change in the distribution of enzymatic activities unrelated to the apical pole of the oxyntic cell. Preliminary, tentative information aimed at identifying the processes responsible for the observed changes is presented.