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1.
- 1. 1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.
- 2. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.
- 3. 3. Among the ligands tested, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate.
- 4. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K
for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.
- 5. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.
Keywords: Ca2+-ATPase; Red cell membrane; Vanadate; Calmodulin 相似文献
2.
Richard Grosse Tom Rapoport Johannes Malur Jrg Fischer Kurt R.H. Repke Ian M. Glynn 《生物化学与生物物理学报:生物膜》1979,550(3)
The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.
- 1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.
- 2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.
- 3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments.
- 4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.
Keywords: (Na+ + K+)-ATPase; SH-group alkylation; Inactivation kinetics; Mathematical modelling; Substrate affinity 相似文献
3.
- 1. (1) VO3− combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca2+-dependent phosphoenzyme.
- 2. (2) VO3− blocks hydrolysis of ATP at the catalytic site. The sites for VO3− also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase.
- 3. (3) The sites for VO3− show positive interactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3−. Although, with less effectiveness, Na+ substitutes for K+ whereas Li+ does not. The apparent affinities for Mg2+ and K+ for inhibition by VO3− seem to be less than those for activation of the Ca2+-ATPase.
- 4. (4) Inhibition by VO3− is independent of Ca2+ at concentrations up to 50 μM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitory effect of VO3−.
Keywords: Ca2+-ATPase; Vanadate inhibition; K+; Li+; (Red cell membrane) 相似文献
4.
5.
Ouabain activation of the phosphatase associated with Na+,K+-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na+ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na+.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg2+-dependent phosphatase and for inhibition of Na+,K+-ATPase and K+-phosphatase. 相似文献
6.
Highly purified Na+, K+-ATPase of the dog kidney was reacted with Mg2++32Pi or Mg2++32Pi + ouabain. 32P-phosphorylation was terminated by the addition of EDTA, and the effects of various ligands on dephosphoration rate were studied. ATP reduced the dephosphorylation rates of both the native and the ouabain-complexed enzymes. K0.5 for this effect of ATP was about 0.2 mM. ADP also slowed dephosphorylation, but less effectively than ATP. The ATP effect on the native enzyme, but not that on the ouabain-complexed enzyme, was antagonized by Na+. The data establish the binding of ATP to the phosphoenzyme. Since the site that is phosphorylated by Pi is the same that is phosphorylated by ATP, coexistence of two ATP sites on the functional unit of the enzyme is suggested. 相似文献
7.
The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (KD) of 13 nM was found in the presence of (Mg2+ + Pi) and (Na+ + Mg2+ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of (Mg2+ + Pi), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of (Na+ + K+)-ATPase. With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献
8.
9.
10.
H. Koepsell 《The Journal of membrane biology》1979,45(1-2):1-20
Summary As different structural states of the (Na+–K+)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na+, K+, Mg++, Pi and ATP on the reaction between highly purified (Na+–K+)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na+–K+)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na+, K+, Mg++ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg++, (ii) Mg++ and Na+ or (iii) Mg++ and K+ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na+, Mg++ and ATP were present together. (c) It was inhibited even more if only (i) Na+, (ii) K+, (iii) ATP or both (iv) ATP and Na+, (v) ATP and K+, (vi) ATP and Mg++, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg++, ATP and K+ were present together.In the presence of Mg++ plus ADP and in the presence of Mg++ plus the ATP analog adenylyl (--methylene) diphosphonate, Na+ and K+ did not influence the degree of antibody inhibition as they did in the presence of Mg++ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg++, ATP and K+ was affected by the sequence in which K+ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na+–K+)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na+ and K+ in the presence of Mg++ plus ATP are proposed to occur during the Na+–K+ transport. 相似文献
11.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(2):365-379
Ouabain-binding and phosphorylation of (Na+ + K+-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitve to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex.Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptice digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1 : 1 stoichiometric relation between this phosphorylation and the amount of bound ouabain.Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of seperate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity. 相似文献
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13.
A method is described for the extraction of microsomal ouabain-sensitive (Na+ + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ + K+)-stimulated activity in the range of 30–40 nmol · mg−1 · min−1 at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+.The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump. 相似文献
14.
(1) The fluorescence of eosin Y in the presence of (Na+ + K+)-ATPase is enhanced by Mg2+. The enhancement by Mg2+ is larger than that obtained with Na+ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg2+ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na+. (2) The Mg2+-dependent enhancement of fluorescence can be reversed by K+ as well as by ATP. In the presence of Mg2+ + Pi (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg2+ and a low concentation of K+ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg2+ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K+-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per 32P-labelling site in the presence of 5 mM Mg2+ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg2+ + 0.25 Pi, the Kd values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg2+, 150 mM Na+, the Kd values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg2+, the addition of K+ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per 32P-labelling site). With 5 mM Mg2+ + 150 mM K+, the affinities of the two binding sites become identical, at a Kd of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg2+-form to the K+-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na+ in the presence of Mg2+ does not appreciably change the rate of conversion to the K+-form, giving a rate constant of about 110 s?. However, the addition of oligomycin in the presence of Mg2+ + Na+ had a profound effect: the rate of conversion to the K+-form was decreased by a factor of 2000 to about 0.063 s?1. This suggests that the conformation with Mg2+ alone is different from the conformation with Na+ alone. (6) The effects of K+, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per 32P-labelling site are bound to ATP sites. 相似文献
15.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,98(2):229-236
- 1.1. The expected higher gill (Na++K+)-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected
- 2.2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg2+, 50 mM Na+, 2.5 mM K+, pH 7.0 in FW, and 3 mM ATP, 10 mM Mg2+, 100 mM Na+, 10 mM K+, pH 7.5 in BW.
- 3.3. The change of the enzyme activation kinetics by Mg2+, ATP, Na+ and K+ from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity.
- 4.4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.
16.
17.
The inhibitory effect of ouabain on (Na+ + K+)-activated ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na+, Mg2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na+ + K+)-activated ATPase reaction was subsequently started by the addition of a KCl solution. 相似文献
18.
H. Koepsell 《The Journal of membrane biology》1979,48(1):69-94
Summary To distinguish ligand-induced structural states of the (Na+–K+)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na+, K+, Mg++ and ATP. It was found that first the large and then the small polypeptide chain of the (Na+–K+)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na+–K+)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na+–K+)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg++, Na+ and K+ present during trypsin digestion influenced the time course and degree of degradation of the (Na+–K+)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na+–K+)-ATPase was the same, whether Na+ or K+ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na+ or K+ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na+ and K+ also influenced the degradation pattern of the (Na+–K+)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na+ and K+ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg++, Na+ and K+ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na+–K+)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na+–K+)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na+–K+)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na+–K+)-ATPase is regulated by binding of Na+ or K+ to the enzyme-ATP complex. The effects of Na+ and K+ on the (Na+–K+)-ATPase structure are modulated by the ATP binding to high affinity and to low affinity ATP binding sites. 相似文献
19.
- 1. 1. Cu2+ at a concentration of 10−4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc).
- 2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.
- 3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc.
- 4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+.
- 5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.
- 6. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux.
- 7. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain.
- 8. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.
Abbreviations: DTNB; 5; 5′-dithiobis(2-nitrobenzoic acid) 相似文献