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PubMed1997

Effect of propranolol on rat brain synaptosomal Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase.

The beta blocker drug propranolol (PPL) significantly inhibited Na(+)-K(+)-ATPase, Mg(2+)-ATPase and Ca(2+)-ATPase activities in a concentration dependent manner in rat brain synaptosomes. The concentrations required for 50% inhibition (IC50) in the activity of these enzymes were 1.5-1.8 mM. The double-reciprocal plot of ATP-stimulated Na(+)-K(+)-ATPase activity in the presence of PPL showed apparent decrease in K(m) and Vmax and the inhibition was of an uncompetitive type with respect to ATP. The nature of inhibition by PPL of Na(+)-activated Na(+)-K(+)-ATPase activity was of a mixed type showing an increase in Km and decrease in Vmax. Potassium activation kinetics of Na(+)-K(+)-ATPase displayed uncompetitive type of inhibition with PPL since Km and Vmax were decreased. Magnesium activation of Mg(2+)-ATPase showed decrease in Vmax with no apparent change in Km in the presence of PPL. The drug inhibited synaptosomal Ca(2+)-ATPase in an uncompetitive manner. The observed inhibition of synaptosomal ATPases indicates possible alterations in the synaptic transmission by the beta blocker drug PPL.

Adrenergic beta-Antagonists↗
PubMed2004

Age-related changes of NA(+), K(+) - ATPase, Ca(+2) - ATPase and Mg(+2) - ATPase activities in rat brain synaptosomes.

Cerebral metabolism of glucose, one of the determinants of tissue ATP level, is crucial for central nervous system function. The activity of P-type pumps, namely Na(+), K(+) - ATPase, Ca(+2) - ATPase and Mg (+2) - ATPase were examined in brain synaptosomes of 5 - day, 3 - month and 18 - month - old rats to determine if changes in enzyme activity related to aging are potentially associated with alterations in glucose homeostasis. Activities of all the ATPases studied in isolated brain synaptosomes were expressed in micromol of Pi liberated from ATP by 1 mg of synaptosome protein during one hour. Serum glucose concentration was measured by the glucose oxidase method and insulin level was estimated by the RIA. Our results demonstrate that 18 - month - old rats are characterized by hyperglycemia and hyperinsulinemia. Their serum glucose concentration was significantly increased approx. 62.3% and 135.8 % as compared to 3 - month - old rats and 5 - day, newborn rats, respectively. An enormous increase in serum insulin concentration in the old, hyperglycemic rats was observed concomitantly. As a result of these changes the insulin - to - glucose ratio in the old rats was greatly increased approx. (270% and 230%) compared to young, mature and newborn rats. Hyperglycemia and hyperinsulinemia occurring in the old rats, had a different impact on activities of the ATPases tested. Our results have revealed that Na(+), K(+) - ATPase activity remains almost unchanged with age, the activity of Ca(+2) - ATPase decreases, whereas that of Mg(+2) - ATPase increases significantly in old, insulin resistant rats. In conclusion it seems that changes in activity of different P - type pumps may differ with aging and that adaptation of specific ATPases to internal environment alterations is not identical.

Adenosine Triphosphatases↗
PubMed1994

Assembly of Na,K-ATPase alpha-subunit isoforms with Na,K-ATPase beta-subunit isoforms and H,K-ATPase beta-subunit.

cDNA encoding an epitope tag was joined to cDNAs encoding the chicken Na,K-ATPase beta 1 and beta 2 and H,K-ATPase beta-subunits to allow recognition of these beta-subunits with the same monoclonal antibody during assembly assays. cDNAs encoding chicken Na,K-ATPase alpha 1, alpha 2, or alpha 3 and Na,K-ATPase beta 1 or beta 2 or H,K-ATPase beta-subunits were transiently coexpressed in mammalian cells. Subunit assembly was assayed by immune precipitation of alpha-isoforms with a monoclonal antibody to the epitope-tagged beta-subunits. Each of the chicken alpha-isoforms assembled with each of the Na,K-ATPase beta-subunits and the H,K-ATPase beta-subunit. Each of the epitope-tagged beta-subunits also assembled with a Na,K-ATPase/Ca-ATPase chimera that retained only 26 amino acids of the Na,K-ATPase alpha-subunit, demonstrating that all three beta-subunits recognize this same alpha-subunit assembly site.

Amino Acid Sequence↗
PubMed1993

Ion transport ATPases as targets for free radical damage. Protection by an aminosteroid of the Ca2+ pump ATPase and Na+/K+ pump ATPase of human red blood cell membranes.

Preincubation of red blood cell membranes in the presence of ferrous sulfate and EDTA resulted in both a concentration- and time-dependent inhibition of the Na+/K+ pump ATPase, basal Ca2+ pump ATPase, and the calmodulin- (CaM) activated Ca2+ pump ATPase. The IC50 for all three ATPases was approximately 2.5 x 10(-5) M iron. The addition to membranes of ferrous iron and EDTA in an approximately 1:1 ratio resulted in conversion to the ferric iron form in several minutes. However, inhibition of the ion pump ATPases and cross-linking of membrane proteins occurred over the course of several hours. The time course of formation of thiobarbituric acid-reactive substances (TBARS) closely paralleled inhibition of the ion pump ATPases. Inhibition of the ion pump ATPases was prevented by the addition of deferoxamine or superoxide dismutase but not by mannitol, or catalase. Both butylated hydroxytoluene and tirilazad mesylate (U74006F) prevented the formation of TBARS, limited the inhibition of the ion pump ATPases, and reduced cross-linking of membrane proteins. These data may be interpreted to suggest that inhibition of ion pump ATPases in plasma membranes may occur as a result of iron-promoted formation of superoxide and subsequent lipid peroxidation, which can be prevented by free-radical scavengers including butylated hydroxytoluene and U74006F.

Butylated Hydroxytoluene↗
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↗
PubMed1997

Assembly of the chimeric Na+/K+-ATPase and H+/K+-ATPase beta-subunit with the Na+/K+-ATPase alpha-subunit.

Two sets of chimeric beta-subunits were constructed from subunits of Torpedo californica Na+/K+-ATPase and pig gastric H+/K+-ATPase. Five unique restriction sites (SnaBI, EcoRV, MunI, SphI and EcoT22I) were created at equivalent positions of the respective cDNAs and were used as joining points for the construction. One set of chimeras (HxN series) was made by exchanging the 5' portion of the Na+/K+-ATPase beta-subunit cDNA with the corresponding portion of the H+/K+-ATPase beta-subunit cDNA at the respective joining point. Complementary constructs were also prepared (NxH series). In the HxN series, the chimera joined at the SnaBI site formed a stable trypsin resistant complex with the Na+/K+-ATPase alpha-subunit, which was functional with respect to ATP hydrolysis and pump current generation, although the activities were less than those of the complex with the Na+/K+-ATPase beta-subunit. Trypsin resistance decreased for the complex of the chimera joined at the EcoRV site. In the NxH series, the chimeras joined at the SnaBI site and the EcoRV site formed rather trypsin-resistant complexes, but the expressions of the alpha-subunits were below 50% of the control. The chimeras joined at the MunI, SphI and EcoT22I site formed complexes susceptible to tryptic digestion. None of the chimeras in the NxH series were functional. These results suggest that at least two regions of the Na+/K+-ATPase beta-subunit [SnaBI site(Tyr40) to EcoRV site(Ile89) and EcoT22I site(Cys176) to C-terminus)] are involved in stable assembly with the Na+/K+-ATPase alpha-subunit and that the cytoplasmic domain [N-terminus to SnaBI site(Tyr40)] is functionally replaceable with the corresponding domain of the H+/K+-ATPase beta-subunit.

Amino Acid Sequence↗
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↗
PubMed2002

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function.

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.

Adenosine Triphosphate↗
PubMedDate not supplied

Comparative studies on the ATPase-binding sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the use of ATP-analogues.

The effects of ATP-analogues on Ca2+-ATPase and (Na++ K+)-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicated by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At lease in (Na+ + K+)-ATPase Na+ and Mg2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP needs Mg+. Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca2+-ATPase this was concomited by the occlusion of Ca2+ in a stable form. No occlusion of Na+ was observable so far in the (Na+ + K+)-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K+, but activated by high concentrations of Na+. Consequently, despite of the inhibition of (Na+ + K+)-ATPase activity, chromium(III)-ATP supported the Na+ - Na+-exchange reaction in everted red blood cells.

Adenosine Triphosphate↗
PubMed1994

ATPase kinetics for wild-type Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the beta-subunit Thr197-->Ser mutation.

Unisite ATPase kinetic constants were measured for wild-type yeast Saccharomyces cerevisiae F1-ATPase and F1-ATPase with the Thr197-->Ser mutation in the beta subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thr197-->Ser mutation in the yeast F1-ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552-16556]. A unifying hypothesis is that the affinity of F1-ATPase for ADP is altered by azide, oxyanions and the Thr197-->Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild-type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197-->Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197-->Ser mutation as measured by determining the KiADP values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild-type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the KiADP values for ATP hydrolysis under multisite conditions approximately threefold for the wild-type and mutant enzymes and the Thr197-->Ser mutation increases KiADP ninefold. The effect of sulfite on KiADP is additive to the effect of the Thr197-->Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197-->Ser mutation on the biochemistry of F1-ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197-->Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197-->Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP.

Adenosine Diphosphate↗
PubMed1994

[Effects of m-nifedipine on activities of Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase in myocardium and brain of elderly renovascular hypertensive rats].

Clipping the left renal artery induced marked hypertension and hypertrophy of the left ventricle in elderly (24-month-old) rats. m-Nifedipine (m-Nif, ig 20 mg.kg-1, qd x 9 wk) significantly decreased the systolic blood pressure and the weight of the left ventricle in the renovascular hypertensive rats (RVHR). In RVHR, the activities of Na+, K(+)-ATPase and Ca(2+)-ATPase in microsomes of the myocardium and brain were reduced significantly compared with those in sham-operated rats. m-Nif was found to increase the activities of these enzymes of the myocardium and brain in elderly RVHR (P > 0.01). m-Nif 10-1000 mumol.L-1 in vitro elevated the activities of Na+, K(+)-ATPase and Ca(2+)-ATPase in the myocardium and brain of RHVR. On the contrary, the myocardial and cerebral Mg(2+)-ATPase activities were higher in RVHR than in sham-operated rats. m-Nif was shown to depress markedly this enzyme both in vivo and in vitro. These results indicate that m-Nif can improve the activities on Na+, K(+)-ATPase and Ca(2+)-ATPase in the myocardium and brain in elderly RVHR.

Animals↗
PubMed1994

Na(+)-ATPase activity of Na(+),K(+)-ATPase. Reactivity of the E2 form during Na(+)-ATPase turnover.

Based on work of Post et al. (Post, R. L. Toda, G., and Rogers, F.N. (1975) J. Biol, Chem. 250, 691-701), we studied the E2 form reactivity of Na(+),K(+)-ATPase (EC 3.6.1.37) during Na(+)-ATPase turnover by following ATP hydrolysis with and without P(i) and enzyme phosphorylation from P(i) at 20 degrees C. For theoretical calculations we employed the Albers-Post scheme assuming that, even with no K+, E2 exhibits the ATP regulatory site. Using available rate constants the model predicts: (i) without P(i), Na(+)-ATPase displays a single high affinity ATP site but becomes double Michaelian (with high and low ATP affinity) when P(i) is present. (ii) Phosphorylation from P(i) can be detected during Na(+)-ATPase (t 1/2 about 400 ms); the KmP(i) is substantially higher that the KdP(i). (iii) P(i) incorporation is reduced by ATP acting with low affinity; this does not require an increase in the E2-E1 transition rate. (iv) The KmATP of the regulatory site is augmented when [P(i)] increases. The experimental observations, using partially purified pig kidney enzyme, agreed with the predictions. In addition they showed that: (i) extracellular Na+ can prevent P(i) incorporation; this effect is additive with that of ATP but with independent Ki values. (ii) Mg2+ stimulates P(i) incorporation with low affinity (Km of 1.5 mM). (iii) beta, gamma-Methyleneadenosine 5'-triphosphate and palmitoyl-CoA antagonize P(i) inhibition of Na(+)-ATPase. These results agree with a model where the Na(+),K(+)-ATPase and Na(+)-ATPase cycles share most of their intermediate steps and enzyme conformations.

Adenosine Triphosphatases↗
PubMed1997

Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta.

Amino acids N886-A911 of the rat Na+-K+-ATPase alpha3-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit. The chimera (NGH26) was expressed in yeast with the rat Na+-K+ -ATPase beta1-subunit (rbeta1), the rat H+-K+-ATPase beta-subunit (HKbeta), the chimeric beta-subunit NHbeta1 (containing the carboxy-terminal ectodomain of HKbeta), or the chimeric beta-subunit HNbeta1 (containing the carboxy-terminal ectodomain of rbeta1). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKbeta and NHbeta1 rather than with rbeta1 or HNbeta1. Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKbeta or NHbeta1. These results suggest that the sequence Q905-V930 interacts with the HKbeta-subunit on the extracellular side of the cell membrane. The NGH26 + HKbeta complex is less stable than alpha3 + HKbeta when heated and also has a lower binding affinity for ouabain [dissociation constant (Kd) = 63 nM] compared with alpha3 + rbeta1 or alpha3 + HKbeta (K(d) = 5-10 nM). In contrast, the NGH26+NHbeta1 complex is thermally as stable as alpha3 + rbeta1 complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type. These results indicate that the amino acids Q905-V930 of the rat gastric H+-K+-ATPase alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.

Amino Acid Sequence↗
PubMed1994

Tight nucleotide binding sites and ATPase activities of the Rhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase.

Solubilized Rhodospirillum rubrum RrF1-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF1 with Mg(2+)-ATP or Mg(2+)-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF1. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF1 has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F1-ATPases have faster koff constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg2+ than Ca(2+)-ATPase activity observed with native RrF1, as with epsilon-depleted, activated CF1. An almost complete conversion of both RrF1 and CF1 from Ca(2+)- to Mg(2+)-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF1 and CF1 divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg2+ ions. The RrF1-ATPase activity is 30-fold more sensitive than CF1 to efrapeptin, and completely resistant to either inhibition or stimulation by the CF1 effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF1 a low-affinity, stimulatory site for tentoxin.

Adenine Nucleotides↗
PubMed2001

Modulation of Na, K-ATPase activity by immunoglobulins--III. Influence of an ATPase activity regulating agent "Marina" on Na, K-ATPase activity in patients with various chronic disturbances.

BACKGROUND: In some earlier work we have demonstrated that hypotonic mineral water can change the functional activity of Na, K-ATPase both in vitro and in vivo. The current investigation was aimed at measuring of the functional activity of ATPase in erythrocytes from blood samples of patients before and after treatment with the artificially mineralised water "Marina". METHODS: "Marina"--an artificial mineral water containing K+ and Mg2+ ions, was investigated by double-blind method. Treatment lasted 7 days, daily intake of "Marina" was 4 x 200 ml before meals. Na, K-ATPase activity in erythrocytes and a number of clinical indices was determined before and after the treatment. RESULTS: After the intake of the preparation "Marina" the normal activity pattern of the Na, K-ATPase in chronic active hepatitis or atherosclerosis patients was restored. The restoration of the Na, K-ATPase activity brought about the normalization of vital clinical indices. CONCLUSIONS: The summarized data from the in vitro and in vivo experiments point to formation of Na, K-ATPase and immunoglobulin complexes as possible cause of cell disfunction and source of organic disturbances.

Arteriosclerosis↗
PubMed2002

The gamma-Na+,K+-ATPase subunit assembles selectively with alpha1/beta1-Na+,K+-ATPase but not with the colonic H+,K+-ATPase.

BACKGROUND: The ubiquitous Na+-pump (Na+,K+-ATPase) assembles as a heterodimer of composition alpha/beta in some nephron segments, while in other segments it may exist as a heterotrimer of composition alpha/beta/gamma. The gamma-subunit has been reported to increase the affinity of the Na+-pump for adenosine 5'-triphosphate (ATP), and decrease affinity for both Na+ and K+. The alpha-subunit of the colonic H+,K+-ATPase (cHK) shares 75% sequence similarity with alpha1-Na+,K+-ATPase (alpha1) and assembles with beta1-Na+,K+-ATPase (beta1) in distal colon and renal medulla. Differences in pharmacological properties have been ascribed to when heterologously expressed function has been compared to function in vitro. The purpose of this study was to determine if cHK might associate with the gamma-subunit of the Na+,K+-ATPase (gamma) as a possible explanation for these variations in function. METHODS: An antibody specific for the gamma was used in coimmunoprecipitation experiments to determine if the gamma assembles stably in vitro with cHK and beta1 in rat renal medulla or distal colon. RESULTS: Our results demonstrate that the gamma-subunit assembles specifically with the Na+-pump, but not with cHK. Furthermore, the gamma-subunit assembly was specific for rat kidney and was not observed in distal colon. CONCLUSION: Since the gamma-subunit did not assemble with the cHK/beta1 complex, gamma-subunit assembly cannot explain those variations in ex vivo and in vitro pharmacologic properties ascribed to cHK.

Animals↗
PubMed1994

Construction, expression and characterization of a plasmid-encoded Na(+)-specific ATPase hybrid consisting of Propionigenium modestum F0-ATPase and Escherichia coli F1-ATPase.

The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon subunits of the H(+)-dependent E. coli ATPase. The transformants showed Na(+)-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na+ and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed.

Adenosine Triphosphatases↗