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1.
Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection 总被引:8,自引:0,他引:8
A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells. 相似文献
2.
HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme. 相似文献
3.
In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered. 相似文献
4.
K Y Lagerspetz K E Senius 《Comparative biochemistry and physiology. B, Comparative biochemistry》1979,62(3):291-293
1. Microsomal preparations from the gills of the freshwater mussel anodonta cygnea cellensis show Mg2+ -dependent Na+ - or K+ -stimulated ATPase activity, which is not inhibited by ouabain. 2. Na+ - or Ka+ -ATPase activity is decreased by Ca2+, acetylcholine, choline, and tetramethylammonium, but slightly increased by ethyl alcohol. 3. It is tentatively suggested that Na+ - or K+ -ATPase is involved in the mechanism of active monovalent cation uptake through the gills of freshwater mussels. 相似文献
5.
Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior.
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Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions. 相似文献
6.
Further biochemical characterization of an Na+ pump inhibitor purified from human urine 总被引:2,自引:0,他引:2
M Crabos M L Grichois P Guicheney I W Wainer J F Cloix 《European journal of biochemistry》1987,162(1):129-135
An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones. 相似文献
7.
Na+-dependent amino acid transport is a major factor determining the rate of (Na+,K+)-ATPase mediated cation transport in intact HeLa cells 总被引:3,自引:0,他引:3
Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane. 相似文献
8.
Adenovirus-dependent increase in cell membrane permeability 总被引:12,自引:0,他引:12
When KB cells were labeled with either 51Cr (1 microCi/ml) or [35S]methionine (5 microCi/ml) and treated with 10 micrograms/ml of adenovirus type 2 (Ad2) at pH 6.0 for 60 min at 37 degrees C, about 25% of the cell-associated 51Cr and 5% of the [35S]methionine were released into the medium. The 51Cr was mainly associated with molecules of 1500 Da or less. When KB cells were labeled with either [3H] choline, alpha-[3H]aminobutyric acid, or [3H]deoxy-2-fluoro-D-glucose and exposed to Ad2, these molecules were released in amounts much higher than 51Cr. The Ad2-dependent release of choline was found to be dependent on Ad2 concentration, with maximum release (nearly 60%) at 10 micrograms/ml of Ad2, on the length of the incubation with Ad2, with maximum release at about 90 min, and on the medium pH with maximum activity at pH 6.0 to 6.5. Greater than 95% of the choline released was water-soluble and identified as choline phosphate. Less than 5% of the choline released was associated with lipids, and none was released as a phospholipid vesicle or micelle. The ability of Ad2 to release choline was abolished by incubating Ad2 for 10 min at 45 degrees C, whereas the binding of Ad2 to the cells was not affected. Fetal calf serum also blocked Ad2-dependent choline release. 相似文献
9.
Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies. 相似文献
10.
Thomas Andersen Schmidt Jim Stenfatt Larsen Keld Kjeldsen 《Journal of neurochemistry》1992,59(6):2094-2104
Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles. 相似文献
11.
Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells. 总被引:6,自引:3,他引:3
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P G Pauw M D Johnson P Moore M Morgan R M Fineman T Kalka J F Ash 《Molecular and cellular biology》1986,6(4):1164-1171
Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17. 相似文献
12.
Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate. 相似文献
13.
G Trigari A R Borgatti A Pagliarani V Ventrella 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,80(1):23-33
Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase. 相似文献
14.
The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration. 相似文献
15.
Zumino Amira Ponce Risler Norma R. Schanne Otto F. Petrich Elena Ruiz Carrión Adriana 《Molecular and cellular biochemistry》1997,169(1-2):85-93
Myocardial Na+,K+-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K+- and Ca2+-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K+-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K+ and Ca2+-ATPases were quantified by K+- and Ca2+-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K+- and Ca2+-dependent pNPPase activities (mol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K+- and Ca2+-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K+- and Ca2+-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K+- and Ca2+-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K+-ATPase concentration and in rodents with reduced Na+,K+- and Ca2+-ATPase concentrations. This may be of importance for development of heart f in hypertrophic heart disease. 相似文献
16.
Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group 总被引:1,自引:0,他引:1
M D Forgac 《The Journal of biological chemistry》1980,255(4):1547-1553
A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent dephosphorylation of the enzyme without inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation. Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of enzyme labeled with the fluorescent sulfhydryl reagent, 2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+ inhibition of activity, the affinity for Mg2+ as an inducing agent for this effect was significantly reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced in magnitude by ouabain and prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+ (and cations that substitute for K+ in supporting activity) induced a 3 to 4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and ATP, although the K+ and Mg2+ effects appeared to be different on the basis of their excitation spectra. The K+ effect was inhibited by ouabain and occurred with a rate greater than the rate of turnover of the enzyme, permitting its involvement in the catalytic cycle. 相似文献
17.
Veklich TO Shkrabak OA Kosterin SO Rodik RV Cherenok SO Boĭko SO Boĭko VI Kal'chenko VI 《Ukrainski? biokhimicheski? zhurnal》2006,78(6):53-63
In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the calix[4]arenes C-97 and C-107 (codes are shown) on ouabain effect on the Na+,K+-ATPase activity. It was shown that calixarenes in concentration 100 tiM inhibited by 97-98% the enzymatic Na+,K+-ATPase activity, while they did not practically influence on the basal Mg2+-ATPase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was < 0.1 microM while for ouabain it was 15-25 microM. The negative cooperative effect was typical of the inhibitory action of calixarenes, as well as ouabain: the value of Hills factor nH = 0.3-0.5 <1. The modelling compound M-3 (0.1 microM 4 microM)--a fragment of the calixarene C-107--did not practically influence the enzymatic activities as Na+,K+-ATPase and basal Mg2+-ATPase. Hence the influence of calixarene C-107 on the Na+, K+-ATPase activity is caused by cooperative action of two fragments M-3 and effect of calixarene bowl, rather than by simple action of the fragment M-3. Calixarenes C-97 and C-107, used in concentration corresponding to values of I0.5 (40 and 60 nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-ATPase activity in the memrane fraction. Under coaction of ouabain with calixarene C-97 or C-107 there was no additive effect of the action of these inhibitors on the Na+,K+-ATPase activity. Calixarene C-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-ATPase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0 +/- 5.2 microM and 5.3 +/- 0.7 microM. It is concluded, that highly effective inhibitors of the Na+,K+-ATPase activity--calixarenes C-97 and C-107 can enhance the effect of the sodium pump conventional inhibitor--ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene C-97). 相似文献
18.
W J Ball 《Biochemistry》1986,25(22):7155-7162
The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site. 相似文献
19.
A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit. 相似文献
20.
Asano S Matsuda S Hoshina S Sakamoto S Takeguchi N 《The Journal of biological chemistry》1999,274(11):6848-6854
2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase. 相似文献