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PubMed1977

Differences in surface membrane ecto-ATPase and ecto-AMPase in normal and malignant cells. I. Decrease in ecto-ATPase in myeloid leukemic cells and the independent regulation of ecto-ATPase and ecto-AMPase.

The hydrolysis of ATP and AMP by enzymes located on the external side of the plasma membrane (ecto-ATPase and ecto-AMPase) was studied in mouse myeloid leukemic cells, normal early myeloid cells, and normal mature granulocytes and macrophages. Nine clones of myeloid leukemic cells were used belonging to three groups that differ in their ability to be induced to differentiate by the differentiation-inducing protein MGI. These three groups consisted of MGI+D+ that can be induced to undergo complete differentiation, MGI+D- that can be induced to partially differentiate and MGI-D- with no induction of differentiation. The ecto-ATPase activity of normal early myeloid cells was similar to that of normal mature granulocytes and macrophages and higher than that of any of the leukemic cells. Among the leukemic cells, the MGI-D- cells had the highest level of ecto-ATPase activity. The behaviour of ecto-AMPase differed from that of ecto-ATPase. Some MGI-D- clones had a higher ecto-AMPase activity than normal cells and MGI+D- and MGI+D+ cells showed no detectable activity. Neither the ecto-ATP-ase nor ecto-AMPase activities changed after induction of differentiation in normal early myeloid or MGI+D+ leukemic cells. The results indicate that the myeloid leukemic cells had a decreased ability to hydrolyse external ATP, that there can be an independent regulation of ecto-ATPase and ecto-AMPase and that neither of these enzyme activities changed during differentiation.

Adenosine Monophosphate↗
PubMed2002

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability.

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.

Adenosine Triphosphatases↗
PubMed1982

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase.

(1) The natural ATPase inhibitor (IF1) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF1 polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF1. The higher the salt concentration, the lower the aggregation state. Aggregation of IF1 was decreased at slightly acidic pH. It increased with the concentration of IF1 as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF1 in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF1 (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547-2551) was shown to have a limited effectiveness as a trap for IF1. The reason was that IF1 released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF1 which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF1 preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336-1344). The particles recovered in the sediment were deprived of IF1 and could therefore be used for preparation of F1-ATPase. The advantage of this method is that both IF1 and F1-ATPase can be prepared from the same batch of mitochondria.

Adenosine Triphosphatases↗
PubMed1979

Binding of ouabain to Na+, K+-dependent ATPase during the ATPase reaction. Evidence for a dimer structure of the ATPase.

Na+, K+-dependent ATPase [EC 3.6.1.3] was purified from porcine kidney by the method of Lane et al. [(1973) J. Biol. Chem. 248, 7197-7200] with slight modifications [Yamaguchi, M. & Tonomura, Y., (1979) J. Biochem. 86, 509-523]. The amounts of a phosphorylated intermediate (EP) and ouabain bound to the enzyme during the ATPase reaction were measured in 2.1 mM MgCl2 and various concentrations of NaCl and KCl at pH 7.5 and 20 degrees C. In presence of NaCl and the absence of KCl, the molar ratio of the amounts of EP and bound ouabain was 1 : 2. In the presence of both NaCl and KCl, it was 1 : 1. In both cases, the amount of bound ouabain was equal to that of EP in the absence of ouabain. These findings suggest that the functional unit of the transport ATPase is a dimer.

Animals↗
PubMed1996

Characteristics of Na+/K(+)-ATPase mediated proton current in Na(+)- and K(+)-free extracellular solutions. Indications for kinetic similarities between H+/K(+)-ATPase and Na+/K(+)-ATPase.

The Na+/K(+)-ATPase of an ouabain-resistant mutant of Torpedo californica and of rat was expressed in Xenopus oocytes by microinjection of mRNA coding for the alpha- and the beta-subunit of the protein. Electrophysiological measurements were performed by means of the extracellular giant-patch clamp technique. The pump currents were analyzed in nominal absence of extracellular Na+ and K+ ions. Under these conditions strongly inward rectifying, ouabain-sensitive current was detected with reversal potentials depending on extracellular pH. Presence or absence of intracellular Na+ or K+ ions had nearly no effect on the inward currents, removal of intracellular ATP caused their inhibition. The reversal potentials of the currents generated by the rat pump was shifted by 82.7 +/- 5.4 mV per 10-fold increase of extracellular proton concentration. This refers to an effective valency of 0.71 +/- 0.05. In the absence of a transmembrane proton gradient the pump current reversed at -64.2 +/- 4.4 mV. These findings are not compatible with a proton conducting channel formed by the pump molecule (Wang and Horisberger (1995) Am. J. Physiol. CP 37, C590-595). Therefore, a kinetic model based on the Post-Albers scheme with a modification derived from the reaction scheme of the gastric H+/K(+)-ATPase is proposed. Together with voltage-dependent binding of extracellular protons, this model is compatible with the observe pump currents.

Adenosine Triphosphate↗
PubMed1990

The bacterial Kdp K(+)-ATPase and its relation to other transport ATPases, such as the Na+/K(+)- and Ca2(+)-ATPases in higher organisms.

The Kdp system is a three-subunit member of the E1-E2 family of transport ATPases. There is sequence homology of the 72 kDa KdpB protein, the largest subunit of Kdp, with the other members of this family. The predicted structure of the 21 kDa KdpC subunit resembles that of the beta subunit of the Na+,K(+)-ATPase, suggesting that these subunits may have a similar function. The 59 kDa KdpA subunit has no known homologue; it is very hydrophobic and is predicted to cross the membrane 10-12 times. Genetic studies implicate this subunit in the binding of K+. As the binding site must be close to the beginning of the transmembrane channel, we suggest that KdpA also forms most or all of the latter. KdpA may have evolved from a K+/H+ antiporter that was recruited by the KdpB precursor to achieve the high affinity and specificity for K+, and the activation of transport by low turgor pressure characteristic of Kdp. Turgor pressure controls the expression of Kdp. This action is dependent on the 70 kDa KdpD and 23 kDa KdpE proteins. We are in the process of sequencing these genes. KdpE is homologous to the smaller protein of other members of a family of pairs of regulatory proteins implicated in control of a variety of bacterial processes such as porin synthesis, phosphate regulon expression, nitrogen metabolism, chemotaxis and nodule formation.

Adenosine Triphosphatases↗
PubMed1984

Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the Ca2+-ATPase of sarcoplasmic reticulum.

We have determined the DNA sequences of the genes encoding the three structural proteins of the Kdp-ATPase, an ATP-driven potassium transport system of Escherichia coli. Regions of the predicted amino acid sequence of KdpB, the phosphorylated protein of the system, are homologous to regions of the Ca2+-ATPase of rabbit sarcoplasmic reticulum. The phosphorylated aspartate residue of the latter is within a region of homology.

Adenosine Triphosphatases↗
PubMed1987

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase.

In order to gain some information regarding Ca2+-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca2+-ATPase activity of myosin purified from rat heart. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by Ca2+ but the maximal activation of Ca2+-dependent ATPase required 4 mM Ca2+ whereas that of myosin ATPase required 10 mM Ca2+. These ATPases were also activated by other divalent cations in the order of Ca2+ greater than Mn2+ greater than Sr2+ greater than Br2+ greater than Mg2+; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca2+-dependent ATPase was not activated by actin. The pH optima of Ca2+-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na+ markedly inhibited Ca2+-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca2+-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca2+-dependent ATPase. Both Ca2+-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH4-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca2+-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3-4 mM Ca2+ and 3 to 4 mM ATP like sarcolemmal Ca2+-dependent ATPase. K+ stimulated both ATPase activities in the absence of Ca2+ and inhibited in the presence of Ca2+. Both enzymes were inhibited by Na+, Mg2+, La3+, and azide similarly. However, Ca2+ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca2+ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca2+-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca2+-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca2+-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca2+-ATPase. These observations suggest that Ca2+-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.

Adenosine Triphosphatases↗
PubMed1996

[Molecular and functional diversity of NA,K-ATPase and renal H,K-ATPases].

Potassium homeostasis is a determinant factor in the maintenance of many vital functions. Cell excitability, for instance, in striate and cardiac muscle, as well as in neurons, is dependent upon the ratio of potassium levels on either side of the plasmic membrane. Acute or chronic mechanisms of adjustment to disorders of bodily potassium balance exist in muscle, the kidney and distal colon. Na+K(+)-ATPase is involved in potassium transfers between the extracellular and intracellular compartments, in particular in muscle, enabling the creation of an appropriate trans-membrane K gradient. Na+K(+)-ATPase also participates in the development and maintenance of a transmembrane potassium electrochemical gradient necessary for potassium secretion processes in the kidney or distal colon. Colonic and renal H+K(+)-ATPases, so-called non-gastric H+K(+)-ATPases, are involved in the absorption of potassium from the gastrointestinal lumen or urinary fluid. They have an important role to play during chronic disorders, e.g. chronic bodily potassium depletion. Renal H+K(+)-ATPases and Na+K-ATPase are P-ATPases, consisting of a heterodimer of two alpha and beta sub-units. Several isoforms have been identified, on both a molecular and functional basis, for both the alpha and beta sub-unit. These two ATPases form part of the Na+K(+)-ATPase/H+K(+)-ATPase gene group. These pumps share many structural and functional similarities, but also particular functional specificities, probably involved in separate physiological roles for each isoform. Four isoforms of the alpha sub-unit and two isoforms of the beta sub-unit of Na+K(+)-ATPase have been identified. Sensitivity to ouabain, a Na+K(+)-ATPase inhibitor, differs according to the alpha isoform present in the alpha beta heterodimer. It is also involved in the catalytic cycle and influences pump potassium affinity. Several H+K(+)-ATPases have been identified from a molecular standpoint: gastric H+K(+)-ATPases and a colonic H+K(+)-ATPase found more recently. Recent studies have shown that both these H+K(+)-ATPases exist in the kidney. "Gastric" H+K(+)-ATPase is active along the entire length of the collecting tubule, in rats exposed to a normal potassium intake. In contrast, colonic H+K(+)-ATPase is active only in the cells of the external medullary collecting duct. This activity cannot be detected in animals on a standard diet but is very powerfully induced by potassium depletion. Activity is independent of steroidal status and of aldosterone in particular. Identification of a molecular homologue in the bladder of the amphibian Bufo marinus (the functional equivalent of the cortical collecting duct of mammals) has enabled the development of functional tests by activity in the oocyte of Xenopus laevis. The use this functional approach has shown that bladder H+K(+)-ATPase, just like that of rat distal colon, is sensitive to ouabain, an inhibitor considered up to now to be specific to Na+K(+)-ATPase. In contrast, this H+K(+)-ATPase shows little or no sensitivity to Sch 28080, a "classical" gastric H+K(+)-ATPase inhibitor. It thus seems that two H+K(+)-ATPases, different from a molecular standpoint, exist in rat kidney. They differ in terms of their cellular activity, regulation and functional properties. This is strongly suggestive of a specific role of each of them in potassium homeostasis, a role which remains to be defined. The use of genetically modified animals, as well as of physiological studies more focussed on this question, should provide clarification of the specific functional role of each isoform of the alpha and beta sub-units of renal H+K(+)-ATPases and Na+K(+)-ATPase. Extrapolation of these results to human pathophysiology is quite another challenge. Control of Na+K(+)-ATPase activity by endoouabain and its effects on cardiovascular pathophysiology must be identified. An H+K(+)-ATPase with molecular and functional characteristics similar to those of amphibian bladder and rat colon H+K(+)-A

Animals↗
PubMed1990

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

Amino Acid Sequence↗
PubMed1985

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products.

Two distinct membrane fractions containing H(+)-ATPase activity were prepared from red beet. One fraction contained a H(+)-ATPase activity that was inhibited by NO(3) (-) while the other contained a H(+)-ATPase inhibited by vanadate. We have previously proposed that these H(+)-ATPases are associated with tonoplast (NO(3) (-)-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP(2-), and Mg(2+) concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg(2+)) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP(2-) in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP(2-) complex.The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H(+)-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg(2+) could modulate ATPase activity at both the tonoplast and plasma membrane.

Journal Article↗
PubMed1996

Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity.

The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.

Adenosine Triphosphatases↗
PubMed1986

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution.

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.

Animals↗
PubMed2006

H+-ATPase activity in selective disruption of H+-K+-ATPase alpha 1 gene of mice under normal and K-depleted conditions.

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H(+)-ATPase and H(+)-K(+)-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAalpha1 (gastric H(+)-K(+)-ATPase) activity is the essential H(+)-K(+)-ATPase activity under normal conditions, and that HKAalpha2 (colonic H(+)-K(+)-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions. To better understand the role of H(+)-ATPase (potassium-independent) in acid secretion and the relationship between H(+)-ATPase and a specific HKA isoform, we examined H(+)-ATPase activity in the H(+)-K(+)-ATPasealpha1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH(i)) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H(+)-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na(+) and K(+), the H(+)-ATPase-mediate pH(i) recovery rates were 6.7 +/- 1.1 x 10(-4) units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 +/- 1.8 x 10(-4) (P < 0.05; n = 6) in HKAalpha1 KO mice. K-independent proton transport activity was significantly inhibited by the H(+)-ATPase inhibitor bafilomycin A(1) (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H(+)-ATPase activity in KO mice. To determine the intracellular localization of H(+)-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H(+)-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H(+)-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H(+)-ATPase-mediate pH(i) recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H(+)-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H(+)-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H(+)-ATPase plays an important role in compensatory proton secretion in the HKAalpha1 KO mice under normal conditions.

Animals↗
PubMed1981

Selective disaggregation of the H+-translocating ATPase. Isolation of two discrete complexes of the rutamycin-insensitive ATPase differing in mitochondrial membrane-binding properties.

The H+-translocating ATPase from rat liver mitochondria can be disaggregated selectively to yield two distinct, stable complexes of the rutamycin-insensitive ATPase. The two ATPase complexes can be purified to homogeneity by zone sedimentation in a glycerol gradient. Based on their electrophoretic mobility in 5% polyacrylamide gels, the aggregates have been designated as type I (Rf = 0.49) ATPase and type II (Rf = 0.56) ATPase. These two complexes of the ATPase differ in ATP hydrolytic activity, in stability, in mobility on 5% polyacrylamide gel electrophoresis, in subunit composition, and in ability to reassociate with submitochondrial particles which are highly depleted in ATPase activity. The type II ATPase is similar to the F1-ATPase, but the type I ATPase contains a 26.5-kilodalton subunit not present in the type II enzyme. This 26.5-kilodalton subunit is equimolar with the gamma subunit of the ATPase (based on Coomassie blue dye binding); its presence seems to be correlated to the altered properties of the type I ATPase. Type I ATPase reconstitutes rutamycin-sensitive ATPase activity in submitochondrial particles treated with trypsin, urea, ammonia, and 1.5% silicotungstic acid. The type II ATPase does not reconstitute rutamycin-sensitive ATPase activity in these ATPase-depleted submitochondrial particles unless it is supplemented with the 26.5-kilodalton subunit isolated from the type I ATPase. The 26.5-kilodalton protein has thus been functionally identified as important for the binding of the ATPase to the membrane by providing a direct link to the membrane or by binding to the ATPase putting it in an appropriate conformation for binding.

Adenosine Triphosphatases↗
PubMed2006

P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti.

This study describes the expression patterns of P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase in the larval and adult forms of the mosquito Aedes aegypti and provides insight into their relative importance in ion transport function of key osmoregulatory organs. RT-PCR assays indicate that, at the level of the gene, both ATPases are expressed in all of the osmoregulatory tissues of larvae (midgut, Malpighian tubules, rectum and anal papillae) and adults (stomach, Malpighian tubules, anterior hindgut and rectum). Immunohistochemical studies determined that both ATPases are present in high levels in all the relevant organs, with the exception of the larval rectum (P-type Na(+)/K(+)-ATPase only). In larval gastric caeca, ATPase location corresponds to the secretory (basal P-type Na(+)/K(+)-ATPase, apical V-type H(+)-ATPase) and ion-transporting (V-type H(+)-ATPase on both membranes) regions as previously described. The two ATPases switch membrane location along the length of the larval midgut, indicating three possible regionalizations, whereas the adult stomach has uniform expression of basolateral P-type Na(+)/K(+)-ATPase and apical V-type H(+)-ATPase in each cell. In both larval and adult Malpighian tubules, the distal principal cells exhibit high expression levels of V-type H(+)-ATPase (apically and cytoplasmically) whereas P-type Na(+)/K(+)-ATPase is highly expressed in stellate cells found only in the distal two-thirds of each tubule. By contrast, the proximal principal cells express both P-type Na(+)/K(+)-ATPase (basal) and V-type H(+)-ATPase (apical). These results suggest a functional segregation along the length of the Malpighian tubules based on cell type and region. P-type Na(+)/K(+)-ATPase is the only pump apparent in the larval rectum whereas in the larval anal papillae and the adult hindgut (including the anterior hindgut and rectum with rectal pads), P-type Na(+)/K(+)-ATPase and V-type H(+)-ATPase localize to the basal and apical membranes, respectively. We discuss our findings in light of previous physiological and morphological studies and re-examine our current models of ion transport in these two developmental stages of mosquitoes that cope with disparate osmoregulatory challenges.

Aedes↗
PubMed2001

Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na(+)/K(+)-ATPase.

In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H(+)-ATPase) is found on the apical membrane of pavement and chloride (Na(+)/K(+)-ATPase-rich) cells, and is an important transporter for energizing Na(+) uptake and H(+) excretion. In the gills of elasmobranch fishes, the V-H(+)-ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H(+)-ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina) and determine if V-H(+)-ATPase and Na(+)/K(+)-ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H(+)-ATPase and greatest number of V-H(+)-ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H(+)-ATPase abundance and the number of V-H(+)-ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H(+)-ATPase and least number of V-H(+)-ATPase-rich cells. In contrast to teleost fishes, localization of V-H(+)-ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H(+)-ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H(+)-ATPase and Na(+)/K(+)-ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid-base regulation where the V-H(+)-ATPase-rich cells and Na(+)/K(+)-ATPase-rich cells are the sites of Cl(-) uptake/HCO(3)(-) excretion and Na(+) uptake/H(+) excretion, respectively.

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