PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “ATPase”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1993

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.

Amino Acid Sequence↗
PubMed2003

[Effect of interleukin-2 on the activity of Ca2+ ATPase and Na+/K+ ATPase of sarcoplasmic reticulum and sarcolemma].

The purpose of the present study was to investigate whether interleukin-2 (IL-2) changes the activity of sarcoplasmic reticulum (SR) Ca(2+) ATPase, sarcolemmal Ca(2+)ATPase and Na(+)/K(+) ATPase by measuring the Pi liberated from ATP hydrolysis with colorimetrical methods. It was shown that the activity of Ca(2+)ATPase in SR from IL-2-perfused (10, 40, 200, 800 U/ml) rat heart increased dose-dependently. After incubation of the SR with ATP (0.1 approximately 4 mmol/L), the activity of SR Ca(2+)ATPase increased dose-dependently in the control group. In the SR from 200 U/ml IL-2-perfused hearts, the activity of Ca(2+)ATPase was much higher than that in the control group. On the other hand, incubation of the SR with Ca(2+) (1 approximately 40 micromol/L) increased the activity of SR Ca(2+) ATPase in the control group. The activity of SR Ca(2+)ATPase of IL-2-perfused hearts was inhibited as the function to Ca(2+). Pretreatment with specific kappa-opioid receptor antagonist nor-BNI (10 nmol/L) for 5 min attenuated the effect of IL-2 (200 U/ml) on the activity of SR Ca(2+) ATPase. After pretreatment with pertussis toxin (PTX, 5 mg/L) or U73122 (5 micromol/L), IL-2 failed to increase SR Ca(2+)ATPase activity. The activity of SR Ca(2+)ATPase was not changed by incubation of SR isolated from normal hearts with IL-2. Perfusion of rat heart with IL-2 did not affect the activity of sarcolemmal Ca(2+)ATPase and Na(+)/K(+)ATPase. It is concluded that perfusion of rat heart with IL-2 increases the activity of SR Ca(2+)ATPase dose-dependently, which is mainly mediated by cardiac kappa-opioid receptor pathway including a PTX sensitive Gi-protein and phospholipase C. IL-2 increases the activity of SR Ca(2+)ATPase as the function to ATP, but inhibits the activity of SR Ca(2+)ATPase as the function to Ca(2+). IL-2 has no effect on the activity of sarcolemmal Ca(2+)ATPase and Na(+)/K(+)ATPase.

Animals↗
PubMed2000

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban.

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells (Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1.Ca to E1'.Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu(37) is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37 degrees C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity ( approximately 0.75 micromol. nmol Ca-ATPase(-1).min(-1) at 15 microM Ca(2+)), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0 degrees C, using 1 microM MgATP, the K(Ca) values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37 degrees C. Measurements of the time course of phosphoenzyme formation at 0 degrees C, using 1 microM MgATP and 268 nM ionized [Ca(2+)], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca(2+) ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E.Ca to E'.Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca(2+) binding to the E1 intermediate.

Adenosine Triphosphate↗
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↗
PubMed1998

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

We previously have demonstrated that the colonic P-ATPase alpha subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H, K-ATPase would be Na+ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na+ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na+ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na+ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a beta subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na+ activity persists when diffusive Na+ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na+ channel, suggesting that the decrease is related to increased active Na+ efflux. The Na+ decrease depends on the presence of K+ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na+, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.

Animals↗
PubMed1999

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes.

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.

Animals↗
PubMed1988

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit.

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.

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↗
PubMed1993

Deficiency of Na+/K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients.

Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.

Adult↗
PubMed2000

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon.

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.

Animals↗
PubMed1987

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase.

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.

Animals↗
PubMedDate not supplied

Na,K-ATPase and Ca-ATPase activities of the myocardial sarcolemma in aging rats after aorta coarctation: role of invertors.

In this work we used aorta coarctation as a model of myocardial hypertrophy. We studied the role of intracellular regulators of plasma membrane status-invertors-in the mechanisms of changes of membrane enzyme activities in the emergency stage of myocardial hypertrophy. We used Wistar rats of various ages: adult (6-8 months) and old (26-28 months) rats. It was shown that 4-6 days after aorta coarctation, in adult rats the activities of both Na,K-ATPase and Ca-ATPase of the sarcolemma of cardiomyocytes increased, but in old rats only the Ca-ATPase activity. Experiments with cell hybrids (cytosol of experimental rats and isolated sarcolemma of cardiomyocytes of intact rats) revealed that cytosol of cardiomyocytes of adult animals after aorta coarctation activated Na, K-ATPase and Ca-ATPase. Cytosol of old intact animals after aorta coarctation did not activate Na,K-ATPase, but activated Ca-ATPase. It was supposed that 4-6 days after aorta coarctation, intracellular regulators (invertors) activating Na,K-ATPase and Ca-ATPase of rat sarcolemma were synthesized in cytosol of adult animals. Invertors activating Na,K-ATPase did not appear after the aorta coarctation in old animals, but factors activating Ca2+-ATPase appeared. Cytosol of adult experimental rats activated Na,K-ATPase of sarcolemmas of cardiomyocytes of intact old animals. The data proved the ability of Na,K-ATPase of sarcolemma of old animals to respond to regulating factors. Based on the divergence between the results of experiments with the Na,K-ATPase and Ca-ATPase activities in old rats, it can be supposed that we were dealing with two different invertors.

Aging↗
PubMedDate not supplied

Ca(2+)-ATPase and Mg(2+)-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes.

In rat jejunal brush-border membranes (BBM), ATP hydrolysis activity was specifically stimulated by CaCl2 and by MgCl2, allowing to identify Ca(2+)-ATPase and Mg(2+)-ATPase activities with a broad pH optimum near 8.0. Nonspecific ATPase activity (in the absence of cations) had a pH optimum above 9.5 as alkaline phosphatase. The effects of Ca2+ and Mg2+ concentrations on ATPase activity evidenced two apparent KA for each cation. At high concentrations, a similar affinity for both cations was recorded (KA: 0.35 mM). At low concentrations, the affinity for Mg2+ was greater than for Ca2+ (KA: 0.02 mM and 0.07 mM respectively). In an attempt to differentially solubilize alkaline phosphatase and ATPase activities, eleven different detergents were assayed. They more or less successfully released Ca(2+)-ATPase and Mg(2+)-ATPase activities from BBM but the more membranes were solubilized by a detergent, the more activities were lost, suggesting a close dependence on integration in BBM. As to alkaline phosphatase and nonspecific ATPase, they almost co-solubilized with Ca(2+)-ATPase and Mg(2+)-ATPase but their total activity was little affected. After treatment of BBM with phosphatidylinositol-specific phospholipase C (E.C. 3.1.4.10), 58% of alkaline phosphatase activity and 45% of nonspecific ATPase activity were released in the supernatant while Ca(2+)-ATPase and Mg(2+)-ATPase activities remained totally incorporated in BBM pellets. These last results definitively demonstrate that Ca(2+)-ATPase and Mg(2+)-ATPase activities are not manifestations of alkaline phosphatase, as earlier suggested, but rather result from the existence of one or several intrinsic membrane enzymes.

Alkaline Phosphatase↗
PubMed2000

High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase.

Na(+),K(+)-ATPase and gastric H(+),K(+)-ATPase are two related enzymes that are responsible for active cation transport. Na(+), K(+)-ATPase activity is inhibited specifically by ouabain, whereas H(+),K(+)-ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na(+),K(+)-ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the alpha-subunit of H(+),K(+)-ATPase were replaced by their counterparts of the alpha(1)-subunit of rat Na(+),K(+)-ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H(+), K(+)-ATPase were replaced by those of Na(+),K(+)-ATPase could form a phosphorylated intermediate, but hardly showed a K(+)-stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na(+),K(+)-ATPase were also included in this chimeric ATPase, K(+)-stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34/56, had obtained a high-affinity ouabain-binding site, whereas the rat Na(+), K(+)-ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na(+),K(+)-ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however, originates from H(+),K(+)-ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1/M2 loop of chimera HN34/56 results in a decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and M5/M6 hairpins of Na(+),K(+)-ATPase are present.

Animals↗
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↗