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1.
(1) A membrane fraction enriched in (Na+ + K+)-ATPase (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an (Na+ + K+)-ATPase specific activity of approx. 2 units/mg and was >95% ouabain-sensitive. (2) The (Na+ + K+)-ATPase had a Km for ATP of 0.42 ± 0.04 mM and a pH optimum of 7.0. It was inhibited by ouabain with a Ki of 0.32 ± 0.04 μM. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with NaCl + KCl = 300 mM. (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, theKm for Mg2+ was 0.86 ± 0.10 mM, and at 6 mM ATP, the Km was 1.86 ± 0.44 mM. High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The (Na+ + K+)-ATPase prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.  相似文献   

2.
The ATP/ADP exchange is shown to be a partial reaction of the (H+ + K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5 -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5 = 116 μM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.  相似文献   

3.
Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.  相似文献   

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6.
A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 μM in the presence of 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate in increased by free Mg2+. The inhibition is half maximally reversed by 250 μM epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 μM epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.  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.
(1) Eosin bound to the (Na+ + K+)-ATPase in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32P-labelling site (KD 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+ (+Mg2+). (4) Eosin, like ATP, increases the Na+ relative to K+ affinity (Na+ + K+ = 150 mM) for Na+ activation of hydrolysis and for Na+ protection against inactivation by N-ethylmaleimide. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of Na+ + K+ the system reacts with two different ATP molecules.  相似文献   

9.
The partial purification of (Na+ + K+)-ATPase from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in p-nitrophenylphosphatase activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of (Na+ + K+)-ATPase which can be phosphorylated by reaction with [γ-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the I50 for (Na+ + K+)-ATPase and p-nitrophenylphosphatase inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit Na+ + K+)-ATPase from other tissues such as oligomycin, Ca2+, vanadate, N-ethylmaleimide, p-chloromercuribenzenesulfonic acid (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the (Na+ + K+)-ATPase and p-nitrophenylphosphatase activities. The K+ congeners were relatively more effective in supporting (Na+ + K+)-ATPase compared to p-nitrophenylphosphatase activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of (Na+ + K+)-ATPase activity, p-nitrophenylphosphatase activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.  相似文献   

10.
(Na+ + K+)-dependent ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+-dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration:
Differences between the three preparations in K0.5 for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in K0.5 produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+-sites are oriented extracellularly, as are those of the (Na+ + K+)-dependent ATPase reaction. Dimethyl sulfoxide accentuated Na+-stimulated K+-dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+-sites of the Na+-stimulated pathway.  相似文献   

11.
(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.  相似文献   

12.
The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na+,+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1 · 102M?1 while ATP binds with an apparent Ka = 1.1 · 104M?2 for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is 203.7 ± 15.2 nm. In the presence of ATP a reduction in size is observed.  相似文献   

13.
Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 μM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.  相似文献   

14.
The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba2+, Sr2+ and Ca2+ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were KADPm = 1.4 × 10?5m and KPim = 2.2 × 10?4m for free chromatophores and KADPm = 2.3 × 10?4m and KPim = 5.6 × 10?4m for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles.  相似文献   

15.
The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca2+ + Mg2+)-ATPase, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca2+ + Mg2+)-ATPase had an apparent half saturation constant of 77 ± 31 nM for free calcium, a maximum reaction velocity of 9.9 ± 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity (Ca2+ + Mg2+)-ATPase was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent Km of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity (Ca2+ + Mg2+)-ATPase. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity (Ca2+ + Mg2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. Since the kinetic properties of the high-affinity (Ca2+ + Mg2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase, the high-affinity (Ca2+ + Mg2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.  相似文献   

16.
The Michaelis-Menten parameters, JM and Km of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na+ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, JM, decreased with decrease in extracellular Na+ for both α-aminoisobutyric acid and phenylalanine but the Km for α-aminoisobutyric acid increased markedly as the Na+ concentration fell whereas the Km for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na+-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na+-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na+-independent but it has a high Km and is not additive with the Na+-dependent flux.  相似文献   

17.
(1) A quantitative study has been made of the binding of ouabain to the (Na+ + K+)-ATPase in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain (Na+ + K+)-ATPase will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (Na+ + K+)-ATPase. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the KD) of ouabain to the M. sexta brain (Na+ + K+)-ATPase under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta (Na+ + K+)-ATPase has a higher KD for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta (Na+ + K+)-ATPase can bind ouabain.  相似文献   

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(1) Treatment of (Na+ + K+)-ATPase from rabbit kidney outer medulla with the γ-35S labeled thio-analogue of ATP in the presence of Na+ + Mg2+ and the absence of K+ leads to thiophosphorylation of the enzyme. The Km value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient nH = 2.6. (2) The thio-analogue (Km = 35 μM) can also support overall (Na+ + K+)-ATPase activity, but Vmax at 37°C is only 1.3 γmol · (mg protein)? · h?1 or 0.09% of the specific activity for ATP (Km = 0.43 mM). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, KmADP = 48 μM at 0.1 mM ATP) and is relatively K+-insensitve. The Km for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation.  相似文献   

20.
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