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1.
L Beaugé 《Biochimica et biophysica acta》1978,527(2):472-484
1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected. 相似文献
2.
F M Schuurmans Stekhoven H G Swarts J J de Pont S L Bonting 《Biochimica et biophysica acta》1985,815(1):16-24
A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer. 相似文献
3.
Xu KY 《Biochemical and biophysical research communications》2005,338(4):1669-1677
Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction. 相似文献
4.
H G Swarts H A Zwartjes F M Schuurmans Stekhoven J J de Pont 《Biochimica et biophysica acta》1987,903(3):525-532
In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+. 相似文献
5.
The steady state levels of Na+-dependent phosphoenzyme (E-P) in the (Na+ + K+)-ATPase (EC 3.6.1.3) of rat brain, obtained from a time course study of phosphoenzyme formation at 4 degrees C, were dependent on the concentration of Na+ in the reaction and were maximal in the presence of 64 mM Na+. The plot of phosphoenzyme vs. Na+ concentration gave a curve which on conversion to a double reciprocal plot (1/E-P vs. 1/Na+) gave a line with two breaks, yielding apparently three linear segments. This may be taken to indicate the presence of multiple Na+ sites for the formation of the phosphoenzyme. To test this hypothesis further, the following approach was taken. By making the assumption that the phosphoenzyme may represent bound Na+, it was possible to subject the data to rigorous multiple-site analysis by utilizing steady-state binding equations described by Klotz and Hunston (1971) (Biochemistry 10, 3065-3069), and by Scatchard (1949) (Ann. N.Y. Acad. Sci. 51, 660-672). The analysis of the data by these methods suggests that there may be three non-equivalent Na+ activation sites for the formation of Na+-dependent phosphoenzyme in the (Na+ + K+)-ATPase. The estimated intrinsic association constants (Ka) for activation by Na+ at each of the three sites were 3.4, 0.295, and 0.025 mM-1, respectively. 相似文献
6.
Hans Hebert Peter L. Jørgensen Elisabeth Skriver Arvid B. Maunsbach 《生物化学与生物物理学报:生物膜》1982,689(3):571-574
Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric αβ-unit of the enzyme protein. In phosphate-induced crystals an occupies one unit cell suggesting that interactions between αβ-units can be of importance in the function of the Na+, K+ pump. 相似文献
7.
Crystallization patterns of membrane-bound (Na+ +K+)-ATPase 总被引:6,自引:0,他引:6
Extensive formation of two-dimensional crystals of the proteins of the pure membrane-bound (Na+ +K+)-ATPase is induced during prolonged incubation with vanadate and magnesium. Some membrane crystals are formed in medium containing magnesium and phosphate. Computer-averaged images of the two-dimensional crystals show that the unit cell in vanadate-induced crystals contains a protomeric alpha beta-unit of the enzyme protein. In phosphate-induced crystals an (alpha beta) 2-unit occupies one unit cell suggesting the interactions between alpha beta-units can be of importance in the function of the Na+, K+ pump. 相似文献
8.
Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2. 相似文献
9.
The number of K+ bound to the (Na+ + K+)-ATPase has been measured under equilibrium conditions by a differential-titration technique (Hastings, D.F. (1977) Anal. Biochem. 83, 416-432). 5.1 K+ were bound per 32P-labelling site. The K'D for K+ was dependent on the concentration of choline, which was included to give ionic strength. K'D was 59 +/- 2.5 microM with 97 mM choline, 26 +/-1.9 microM with 30 mM choline. The K+ : choline selectivity was 2564 : 1 and the calculated K'D for K+ with zero choline was 11 microM and for choline with zero K+ was 28 mM. 20 microM ATP in the presence of 97 mM choline incresed the K'D for potassium 3-fold to 177 +/- 14 microM. The K'D for K+ with 3 mM Na+ in the presence of 27 mM choline was 81 +/- 10 microM and with 30 mM Na+ without choline 700 +/- 250 microM. The calculated K'D for Na+ at zero K+ and zero choline was 0.6 +/- 0.2 mM. The K+ : Na+ selectivity was 54 : 1. 相似文献
10.
11.
V. Yu. Tashkin A. A. Shcherbakov H. -J. Apell V. S. Sokolov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(2):113-121
The changes in capacitance and conductance of lipid bilayer membranes have been studied with adsorbed membrane fragments containing Na+,K+-ATPase. These changes have been initiated by fast release of protons from a bound form (“caged H+”) induced by an UV flash. The changes of the capacitance in the presence of Na+,K+-ATPase were affected by the frequency of the applied voltage, pH and the concentration of sodium ions. Addition of sodium ions altered the changes of capacitance caused by a pH jump in the medium due to caged H+ photolysis, and the magnitude and sign of this effect depended on the initial pH. These results are explained by competitive binding of sodium ions and protons to the ion-binding sites of the Na+,K+-ATPase at its cytoplasmic side. The pH at which the sign of the sodium ion effect changed allows the evaluation of the pK of the proton binding site, which is about 7.6. 相似文献
12.
Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate 总被引:1,自引:0,他引:1
Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity. 相似文献
13.
The temperature dependence of (Na+ + K+)-ATPase was measured, utilizing preparations of enzyme from heat and kidney of rats, hamsters, guinea pigs, ground squirrels, turtles, chickens, and ducks. The two hibernating species, hamsters and ground squirrels, were studied awake at normothermia and hibernating at 4 degrees C. The results for every species except the turtles showed the same temperature dependence established for (Na++K+)-ATPase from rabbit kidney with a quasi-linear dependence above 15 degrees C and little or no activity below 15 degrees C. Turtle enzymes showed a broad activity versus temperature curve with a fall-off at high and low temperatures. The data in all cases, including the turtle data, may be fitted by a previously described thermodynamic kinetic model. Further, the model will fith the turnover or decrease in enzyme activity at higher temperatures observed in a number of cases. These results do not support the widely imputed ion pumping role for (Na++K+)-ATPase. 相似文献
14.
W. H. M. Peters A. G. H. Ederveen M. H. L. Salden J. J. H. H. M. de Pont S. L. Bonting 《Journal of bioenergetics and biomembranes》1984,16(3):223-232
Goat antisera against (Na+ + K+)-ATPase and its isolated subunits and against (K+ + H+)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na+ + K+)-ATPase antisera cross-reacted with (K+ + H+)-ATPase or inhibited its enzyme activity. The same was true for the (K+ + H+)-ATPase antiserum with regard to (Na+ + K+)-ATPase and its subunits and its enzyme activity. So not withstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.Number LIII in the series Studies on (Na+ + K+)-Activated ATPase. 相似文献
15.
Showdomycin inhibited pig brain (Na+ + K+)-ATPase with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1) Na+ + Mg2+ + ATP, (2) Mg2+, Mg2+ + K+, K+ and none, (3) Na+ + Mg2+, Na+, K+ + Na+ and Na+ + K+ + Mg2+, (4) Mg2+ + K+ + ATP, K+ + ATP and Mg2+ + ATP, (5) K+ + Na + + ATP, Na+ + ATP, Na+ + K+ + Mg2+ + ATP and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition (KS0.5) were 3 mM, 0.13 mM and 4 MicroM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i. e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of (Na+ + K+)- ATPase proceeds via at least four kinds of enzyme form (E1, E2, E1 . nucleotide and EP), which all have different conformations. 相似文献
16.
Experiments on the effects of varying concentrations of Ca2+ on the Mg2+ + Na+-dependent ATPase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase showed that Ca2+ was a partial inhibitor of this activity. When Ca2+ was added to the reaction mixture instead of Mg2+, there was a ouabain-sensitive Ca2+ + Na+-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg2+ + Na+-dependent activity. The apparent affinities of the enzyme for Ca2+ and CaATP seemed to be higher than those for Mg2+ and MgATP. Addition of K+, along with Ca2+ and Na+, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca2+ + Na+ + K+-dependent ATPase was about two orders of magnitude smaller than that of Mg2+ + Na+ + K+-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca2+, Na+, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K+. The enzyme also exhibited a Ca2+ + Na+-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca2+ on Mg2+-dependent activities, nor the Ca2+-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg2+ in all reaction mixtures, it could not be determined whether the apparent Ca2+-dependent activities involved enzyme-substrate complexes containing Ca2+ as the divalent cation or both Ca2+ and Mg2+. 相似文献
17.
18.
The structural organization of crystalline, membrane-bound (Na+ + K+)-ATPase was studied by negative staining and thin sectioning. The enzyme molecules were induced to form crystalline arrays within fragments of membrane by incubation in defined ionic conditions. The enzyme remained fully active after crystallization. Negative staining and computer processing of images of the crystalline specimens identified two discrete crystalline arrays. The dimensions of the unit cell of one of the arrays were large enough to accommodate an alpha beta protomer; those of the other array, an (alpha beta)2 diprotomer . Thin sections of the crystalline fraction contained a unique membrane complex that was formed from two apposed plasma membranes. The paired membranes in this complex were separated by a center-to-center space of 15 nm containing evenly spaced septa that connected the membrane surfaces; the overall thickness of the entire structure was 22-25 nm. The agglutinin from Ricinus communis, a lectin that binds to the carbohydrate moiety of the beta-subunit of (Na+ + K+)-ATPase, decorated the free surfaces of the complex. Therefore, this complex of paired membranes is the result of interactions between the cytoplasmic domains of the enzyme. From measurements of the dimensions of these structures, we estimate the overall length of the enzyme to be approximately 11.5 nm along the axis perpendicular to the plane of the membrane, and the molecular protrudes more (approximately 5 nm) on the cytoplasmic surface than on the extracytoplasmic surface (approximately 2 nm). 相似文献
19.
Abstract— The activities of (Na+ K+)-ATPase and its proposed partial reactions, K +-pNPPase and Na +-dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na +-dependent phosphorylation. The fraction of total phosphorylation that is Na +-dependent increases steadily during development. suggesting a precursor role for some of the Na +-independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na +-K +)-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg 2+ -ATPase activity. Mg 2+ -paranitrophenylphosphatase activity, and Na+-independent phosphorylation levels. The most rapid changes in (Na+-K+)-ATPase take place during the period corresponding to glial proliferation and neuronal arborization. 相似文献
20.
K Satoh T Nakao F Nagai I Kano A Nakagawa K Ushiyama O Urayama Y Hara M Nakao 《Biochimica et biophysica acta》1989,994(2):104-113
Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles. 相似文献