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1.
Homogeneity or heterogeneity of rat soleus-muscle Na,K-ATPase (Na+ + K+-dependent ATPase) with respect to affinity for [3H]ouabain was evaluated. Since the standard method for measuring specific [3H]ouabain binding to rat skeletal-muscle samples includes subtraction of a value for non-specific [3H]ouabain uptake and retention, and a wash-out in the cold to remove [3H]ouabain from the extracellular phase, it was possible that these procedures could hide a class of [3H]ouabain-binding sites either with low affinity or with a rapid dissociation of [3H]ouabain. However, measurements of [3H]ouabain uptake and retention over the range 0.1-5 mM, as well as the omission of wash-out, gave no evidence for heterogeneity of [3H]ouabain-binding sites in rat soleus muscle. Furthermore, the observation of agreement between the uptake and retention of non-specific [3H]ouabain and of [14C]sucrose gave no evidence for the existence of a major pool of [3H]ouabain-binding sites with low affinity for [3H]ouabain. Assuming homogeneity, the total concentration of [3H]ouabain binding sites in soleus-muscle samples from 12-week-old rats is 278-359 pmol/g wet wt.  相似文献   

2.
Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.  相似文献   

3.
Two functional isoforms (1) and + (3) of the Na,K-ATPase catalytic subunit coexist in canine cardiac myocytes [J. Biol. Chem. (1987) 262, 8941-8943]. The in vitro turnover rates of ATP hydrolysis have been determined in sarcolemma preparations by comparing [3H]ouabain-binding and Na,K-ATPase activity at various doses of ouabain (0.3–300 nM). The correlation between the occupancy of the ouabain-binding sites and the degree of Na,K-ATPase inhibition was not linear. The results showed that the form of low-affinity for ouabain (Kd = 300–700 nM) exhibited a lower turnover rate (88 ± 10 vs. 147 ± 15 molecules of ATP hydrolyzed per second per ouabain-binding site) than the high affinity form (Kd = 1–8 nM). Thus our results indicate this specific isoform kinetic difference could contribute to differences in the cardiac cellular function.  相似文献   

4.
[3H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K+. By utilising high K+ incubations to estimate non-specific [3H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 . 10(-7) M and the total maximum binding to be 2.33 . 10(5) sites/cell. Ouabain inhibition of (Na+, K+)-pump function was monitored by the cellular uptake of 86Rb over 5 min. The larger fraction of 86Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 . 10(-7) M. The cellular distribution of the (Na+ + K+)-ATPase was investigated using [3H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [3H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.  相似文献   

5.
Localization of Na+-pump sites in frog skin   总被引:12,自引:6,他引:6       下载免费PDF全文
The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.  相似文献   

6.
The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.  相似文献   

7.
A centrifugation method has been used for determination of [14C]ADP and [3H]ouabain binding to Na,K-ATPase from pig kidney with high specific activity. In the presence of K+, the fit of the [14C]ADP binding data to a two-site model gives a component with high affinity which accounts for 12 +/- 2% of the total sites. The figure is significantly different from 50%, i.e., two components of equal size cannot be assumed. This contrasts with a ratio between the sites of 1:1 obtained by the rate dialysis technique. The discrepancy may be due to the fact that the centrifugation method enables bound ADP to be determined at lower concentrations of free ligand. [3H]Ouabain binding in the absence of Na+ is compatible with a straight line in a Scatchard plot if the isotope is purified shortly before use. An unspecific binding of ouabain can be neglected if the concentration of free ouabain is not too high. In the presence of Na+, the isotherms become upward concave. An analysis of the binding data gives a 19:81% division, although equilibrium is not quite attained. This is a maximum value because the lack in equilibrium will be most pronounced at the small values of free ouabain. Thus the ADP-binding studies are supported. The finding here is in some agreement with the semiquantitative immunoassay showing that pig kidney enzyme contains the isoenzymes alpha 1, alpha 2 and alpha 3 in a proportion of 84:12:4, respectively. Determination of ADP- and ouabain-binding site stoichiometry favours a theory with one substrate site per (alpha beta)2.  相似文献   

8.
1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.  相似文献   

9.
The influence of age on the number of (Na+K+)-ATPase units in skeletal muscle has been assessed by measurements of [3H]ouabain binding in vitro and in vivo to rat soleus muscle. In vitro measurements showed that from the 2nd to the 28th day of life, the number of [3H]ouabain-binding sites increases from 120 to 580 pmol/g wet wt. This is followed by a decrease, until a plateau between 150 and 200 pmol/g is reached around 150 days after birth. 60 min after intraperitoneal injection of [3H]ouabain (12.5 mumol/kg body weight), the soleus muscles of 28-day-old rats had accumulated 2.4-times more 3H-activity per g wet wt. than muscles of 85-day-old rats and the 3H-activity in plasma was 54% lower. The results may explain the low sensitivity to digitalis glycosides found in infants as compared to premature or adult individuals.  相似文献   

10.
The association and dissociation rate constants for the interaction of [3H]-ouabain with partially purified rat brain (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in vitro were estimated from the time course of the [3H]-ouabain binding observed in the presence of Na+, Mg2+ and ATP by a polynomial approximation-curve-fitting technique. The reduction of the association rate constant by K+ was greater than its reduction of the dissociation rate constant. Thus, the affinity of Na+,K+)-ATPase for ouabain was reduced by K+. The binding-site concentration was unaffected by K+. Consistent with these findings, the addition of KCl to an incubation mixture at the time when [3H]-ouabain binding to (Na+,K+)ATPase is close to equilibrium, caused an immediate decrease in bound ouabain concentration, apparently shifting towards a new, lower equilibrium concentration. Dissociation rate constants which were estimated following the termination of the ouabain-binding reaction were different from those estimated with above methods and may not be useful in predicting the ligand effects on equilibrium of the ouabain-enzyme interaction.  相似文献   

11.
Specific binding of radiolabeled inhibitor was employed to localize the Na-pump sites (Na,K-ATPase) in rectal gland epithelium, a NaCl-secreting osmoregulatory tissue which is particularly rich in pump sites. Slices of gland tissue from spiny dogfish were incubated in suitable [3H]ouabain-containing media and then prepared for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative freeze-dry autoradiography at the light microscope level. Gross freezing or drying artifacts were excluded by comparison with additional aldehyde-fixed slices. Characterization experiments demonstrated high-affinity binding which correlated with Na,K-ATPase inhibition and half-saturated at approximately 5 microM [3H]ouabain. At this concentration, the normal half-loading time was approximately 1 h and low-affinity binding to nonspecific sites was negligible. Autoradiographs from both 1- and 4-h incubated slices showed approximately 85% of the bound [3H]ouabain to be localized within a 1-micrometer wide boundary region where the highly infolded basal-lateral cell membrane are closest to the mitochondria. These results establish that most of the enormous Na,K-ATPase activity associated with rectal gland epithelium is in the basal-lateral cell membrane facing interstitial fluid and not in the luminal membrane facing secreted fluid. Moreover, distribution along the basal-lateral membrane appears to be nonuniform with a higher density of enzyme sites close to mitochondria.  相似文献   

12.
Exercise is associated with a net loss of K+ from the working muscles and an increased plasma K+ concentration, indicating that the capacity for intracellular reaccumulation of K+ is exceeded. Training reduces the exercise-induced rise in plasma K+, and an increased plasma [K+] may interfere with physical performance. Since the clearing of K+ from the extracellular space depends on the capacity for active K+ uptake in skeletal muscle, the effects of training and inactivity on the total concentration of (Na+ + K+)-ATPase was determined. Following 6 weeks of swim training, the concentration of [3H]ouabain-binding sites in rat hindlimb muscles was up to 46% (P less than 0.001) higher than in those obtained from age-matched controls. Whereas muscle Na+, K+ contents remained unchanged, the concentration of citrate synthase increased by up to 76% (P less than 0.001). Training induced no change in the [3H]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0.02-0.001) within 1 week. In conclusion, training leads to a significant and reversible rise in the concentration of (Na+ + K+)-ATPase in muscle cells. This may be of importance for the beneficial effects on physical performance by improving the maximum capacity for K+ clearance.  相似文献   

13.
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   

14.
P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.  相似文献   

15.
Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.  相似文献   

16.
The degree of heterogeneity of active Na+/K(+)-ATPases has been investigated in terms of ouabain sensitivity. A mathematical analysis of the dose-response curves (inhibition of Na+/K(+)-ATPase) at equilibrium is consistent with the putative existence of three inhibitory states for ouabain two of high (very high plus high) and one of low affinity. The computed IC50 values are: 23.0 +/- 0.15 nM, 460 +/- 4.0 nM and 320 +/- 4.6 microM, respectively. The relative abundance of the three inhibitory states was estimated as: 39%, 36% and 20%, respectively. Direct measurements of [3H]ouabain-binding at equilibrium carried out on membrane preparations with ATP, Mg2+ and Na+ also revealed two distinct high affinity-binding sites, the apparent Kd values of which were 17.0 +/- 0.2 nM (very high) and 80 +/- 1 nM (high), respectively. Dissociation processes were studied at different ouabain concentrations according to both reversal of enzyme inhibition and [3H]ouabain release. The reversal of enzyme inhibition occurred at three different rates, depending upon the ouabain doses used (10 nM, 2 and 100 microM). When the high-affinity sites were involved (ouabain doses lower than 2 microM) the dissociation process was biphasic. A similar biphasic pattern was also detected by [3H]ouabain-release. The time-course of [3H]ouabain dissociation (0.1 microM) was also biphasic. These data indicate that the three catalytic subunits of rat brain Na+/K(+)-ATPase alpha 1, alpha 2 and alpha 3 (Hsu, Y.-M. and Guidotti, G. (1989) Biochemistry 28, 569-573) are able to hydrolyse ATP and exhibit different affinities for cardiac glycosides.  相似文献   

17.
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   

18.
Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.  相似文献   

19.
The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the "high-affinity" [3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.  相似文献   

20.
Canine cardiac myocytes contain two distinct molecular forms of the Na,K-ATPase catalytic subunit. They are resolved by gel electrophoresis and identified using immunological techniques. The apparent molecular weights of the catalytic subunits are 95,000 (alpha) and 98,000 (alpha +). As judged by [3H]ouabain-binding measurements and Na,K-ATPase assays, the two forms are active and differ by a factor of 150 in their respective affinity for digitalis (ouabain and digitoxigenin). The dissociation constant of the high affinity form (alpha +) is KD, 2 nM, and that of the low affinity molecular form (alpha) is KD, 300 nM. According to both enzymatic and binding assays, up to 70% of maximum inhibition is caused by occupation of the high affinity sites (alpha +). Inasmuch as the pharmacological and toxic concentrations of digitalis in dog are 1 and 200 nM, respectively, and as maximum inhibition of Na+ pump in vivo should not exceed 80% to avoid toxicity (Akera, T. and Brody, T. (1982) Annu. Rev. Physiol. 44, 375-388), it appears that the high affinity molecular form (alpha +) is the pharmacological receptor exclusively related to positive inotropy, whereas the low affinity form (alpha) is mainly associated with toxicity.  相似文献   

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