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PubMedDate not supplied

Stimulation of canine kidney BBMV ATPase activity by acidic pH in the presence of Zn2+: an ATPase activity distinct from transport ATPases and alkaline phosphatase that may be an ecto-ATPase.

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.

Adenosine Triphosphatases↗
PubMed1988

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase.

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).

Adenosine Triphosphatases↗
PubMed1991

Discrimination of Ca(2+)-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities.

We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca(2+)-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca(2+)-dependent ATPase activity, in spite of having little or no Ca(2+)-induced isometric tension. The residual ATPase activity is ascribed to the Ca(2+)-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca(2+)-dependence of the ATPase activity is very similar to that of Ca(2+)-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca(2+)-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca(2+)-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca(2+)-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca(2+)-ATPase of SR. Ca(2+)-ATPase activity of SR in skinned fibres was inhibited completely by 10 microM CPA and held to one-half by about 0.2 microM. This effect was only partially reversible. CPA at 10 microM or higher concentrations showed Ca(2+)-sensitizing action on myofibrils, which was readily reversible. CPA at 3 microM inhibited almost completely the Ca(2+)-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.

Animals↗
PubMedDate not supplied

Comparison of ATP binding in the active sites of (Na+ + K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase with low affinity to calcium from cardiac sarcolemma.

The chemical composition of the active sites of cardiac sarcolemmal (Na+ + K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase has not been determined definitely. The present study deals with investigation of the role of OH group in position two on the ribose moiety of the ATP molecule in its interaction with the specific ATP binding sites on the above ATPases. Experiments with application of ATP and deoxyATP (the OH group in position 2 on the ribose absent revealed that neither Ca(2+)-ATPase nor Mg(2+)-ATPase is able to distinguish between ATP and deoxyATP as substrates). This indicates that the OH group investigated may play a negligible role only in ATP binding and splitting by the latter ATPases. On the contrary, kinetic studies of Na+ + K(+)-ATPase activation by deoxyATP revealed that the latter compound is a considerably less suitable substrate for the enzyme than ATP. Consequently the OH group in position 2 on the ribose moiety proved to be important both for ATP binding in the active site and for proper substrate turnover by (Na+ + K(+)-ATPase interaction of the ATP binding site of heart sarcolemmal ATPases. Results of the experiments showed that Ca(2+)-ATPase and Mg(2+)-ATPase cannot distinguish between ATP and deoxyATP as substrates. Kinetic studies of (Na+ + K+)-ATPase activation by deoxyATP revealed that the latter is a considerably less good substrate for the enzyme than ATP. It means that the OH group in position two on the ribose moiety proved to be important for both binding of ATP in the active site and for proper substrate turnover by (Na+ + K+)-ATPase.

Adenosine Triphosphatases↗
PubMed1987

Effects of calcium antagonists on (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities of rat cortical synaptosomes.

1. The effects of 11 calcium antagonists on (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities of rat cortical synaptosomes were studied. 2. All the calcium antagonists studied had inhibitory effects on ouabain-sensitive (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities in synaptosomes at high concentrations (10 or 100 microM). 3. Calcium antagonists such as trifluoperazine, flunarizine and cinnarizine had inhibitory effects on Ca2+-ATPase activity at low concentrations (1-10 microM). 4. Trifluoperazine and La3+ had inhibitory effects on Mg2+-ATPase activity at low concentration (1 microM). 5. Our results suggest that most of the calcium antagonists studied have little effects on neuronal (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities at therapeutic dose ranges (1 microM or lower).

Adenosine Triphosphatases↗
PubMed1992

Inhibition of H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase by strophanthidin.

Studies of the effect of strophanthidin on H(+)-transporting ATPase, Ca(2+)-transporting ATPase and H+/K(+)-transporting ATPase activities are reported. Inhibition observations and kinetic results suggest the existence of a common digitalis aglycone binding site located on the extracellular surface of the enzyme, which is affected competitively by the binding of potassium to H(+)-transporting ATPase, Ca(2+)-transporting ATPase, as well as H+/K(+)-transporting ATPase and Na+/K(+)-transporting ATPase. This may lead to a better understanding of the mechanism of the pharmacological action of cardiac glycosides and imply the possibility that the positive inotropic effect may result from the inhibition of both Ca(2+)-transporting ATPase and Na+/K(+)-transporting ATPase.

Animals↗
PubMed1992

Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus.

A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.

Adenosine Triphosphatases↗
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↗
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↗
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↗
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↗
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↗
PubMed1988

AlF4- reversibly inhibits 'P'-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.

Adenosine Triphosphatases↗
PubMed1992

Ouabain-insensitive, vanadate-sensitive K(+)-ATPase of rat distal colon is partly similar to gastric H+,K(+)-ATPase.

A membrane fraction from rat distal colon contained both ouabain-sensitive and -insensitive K(+)-ATPase activities, which were measured under Na(+)-free conditions. About 38% of the ouabain-insensitive K(+)-ATPase activity was inhibited by vanadate. It was determined whether the ouabain-insensitive, vanadate-sensitive K(+)-ATPase in the colon is similar or identical to gastric H+,K(+)-ATPase. This colonic K(+)-ATPase activity was inhibited completely by monoclonal antibody HK4001, which inhibits the hog gastric H+,K(+)-ATPase activity but not Na+,K(+)-ATPase or Ca(2+)-ATPase. The colonic ATPase activity was inhibited partly by SCH 28080, which is a specific reversible inhibitor of gastric H+,K(+)-ATPase. The colonic ATPase activity was stimulated by low concentrations of K+ (its half-maximal stimulating concentration was 1 mM) and inhibited by high concentrations of K+ (its half-maximal inhibiting concentration was 10 mM), indicating that high and low K+ affinity sites are present in the colonic enzyme as in gastric H+,K(+)-ATPase and that this enzyme is not fully operative under normal physiological conditions. Two other monoclonal antibodies, which inhibit the gastric H+,K(+)-ATPase activity, did not inhibit the colonic K(+)-ATPase activity. The present results suggest that the colonic ouabain-insensitive K(+)-ATPase is partly similar but not identical to the gastric H+,K(+)-ATPase.

Adenosine Triphosphatases↗
PubMed1992

The energy transduction mechanism is different among P-type ion-transporting ATPases. Acetyl phosphate causes uncoupling between hydrolysis and ion transport in H+,K(+)-ATPase.

H+,K(+)-ATPase, Na+,K(+)-ATPase, and Ca(2+)-ATPase belong to the P-type ATPase group. Their molecular mechanisms of energy transduction have been thought to be similar until now. Ca(2+)-ATPase and Na+,K(+)-ATPase are phosphorylated from both ATP and acetyl phosphate (ACP) and dephosphorylated, resulting in active ion transport. However, we found that H+,K(+)-ATPase did not transport proton nor K+ when ACP was used as a substrate, resulting in uncoupling between energy and ion transport. ACP bound competitively to the ATP-binding site of H+,K(+)-ATPase. The hydrolysis of ACP by H+,K(+)-ATPase was stimulated by cytosolic K+, the half-maximal stimulating K+ concentration (K0.5) being 2.5 mM, whereas the hydrolysis of ATP was stimulated by luminal K+, the K0.5 being 0.2 mM. Furthermore, during the phosphorylation from ACP in the absence of K+, the fluorescence intensity of H+,K(+)-ATPase labeled with fluorescein isothiocyanate increased, but those of Na+,K(+)-ATPase and Ca(2+)-ATPase decreased. These results indicate that phosphorylated intermediates of H+,K(+)-ATPase formed from ACP are not rich in energy and that there is a striking difference(s) in the mechanism of energy transduction between H+,K(+)-ATPase and other cation-transporting ATPases.

Adenosine Triphosphatases↗
PubMed1990

Human gastric (H+ + K+)-ATPase gene. Similarity to (Na+ + K+)-ATPase genes in exon/intron organization but difference in control region.

The human gastric (H+ + K+)-ATPase gene (15 kilobases) was cloned, and its nucleotide sequence was determined. The gene has 22 exons and codes a protein of 1,035 residues including the initiator methionine (Mr = 114,047). A conserved lysine-rich sequence with inserted glycine residues was found near the amino terminus of the enzyme. The phosphorylation site and pyridoxal 5'-phosphate- and fluorescein isothiocyanate-binding residues found in the rat and pig enzymes are also conserved in the human enzyme. The positions of introns in the human (H+ + K+)-ATPase gene are essentially the same as those in the human (Na+ + K+)-ATPase alpha and alpha III subunits; but the first introns of the two enzymes are difficult to align, and unlike in the (Na+ + K+)-ATPase gene, the sixth exon in the (H+ + K+)-ATPase gene is not separated by an intron. Furthermore, the ninth intron is located two bases upstream of the position for the corresponding intron of the (Na+ + K+)-ATPase alpha III subunit. The similarity in organization of these two ATPase genes and the homology in the primary structures of their proteins (approximately 60%) suggest that these two genes were derived from a common ancestral gene. However, the 5'-flanking regions of the genes for (H+ + K+)-ATPase and the (Na+ + K+)-ATPase alpha (+) subunit show no apparent sequence homology, indicating that their transcriptions are regulated differently. The control region of the fast-twitch sarcoplasmic reticulum Ca2(+)-ATPase gene also showed no sequence homology to that of (H+ + K+)-ATPase. The 5'-flanking region of the (H+ + K+)-ATPase gene contains potential binding sites for RNA polymerase II and various transcriptional regulation factors and several direct and inverted repeat sequences which may be important for specific and controlled expression of the gene in gastric parietal cells. There are two polyadenylation signals in the 3'-flanking region of the (H+ + K+)-ATPase gene, but the sequence of this region shows no homology to those of the corresponding regions of the genes for the (Na+ + K+)-ATPase alpha and alpha III subunits.

Adenosine Triphosphatases↗
PubMed1992

Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.

Adenosine Triphosphatases↗