Large-scale preparation of Na,K-ATPase from ox brain

Klodos, Irena; Ottolenghi, P; Boldyrev, A. A.

doi:10.1016/0003-2697(75)90311-5

Cited by 74 publications

(20 citation statements)

References 11 publications

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“…(Na' + K +)-ATPase was prepared from beef brain [33] with a specific enzymatic activity of 1-3 U/mg or from pig kidney [34] with a specific activity of 8-15 U/mg. At least 97 % of the total enzymatic activity could be inhibited by 0.1 mM ouabain in both enzyme preparations.…”

Section: Enzyme and Assaysmentioning

confidence: 99%

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

1980

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(Na' + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(II1) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na' + K')-ATPase activity was accompanied by a parallel decrease of the associated K +-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na '-dependently, a phosphointermediate from [y-32P]ATP.The kinetics of inactivation and of phosphorylation with [ I > -~~P ] C~A T P and [ u -~~P I C~A T P are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme : ADP E + CrATP e E . C r A T P e E c r f E c r t E + C?' + P,. The dissociation constant of the CrATP complex of the pig kidney enzytne at 37 ' C was 43 pM. The inactivation rate constant (k+Z = 0.033 min-') was in the range of the dissociation rate constant k dof ADP from the enzyme of 0.011 min-'. The phosphoenzyme was unreactive towards ADP as well as to K + . N o hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na' reactivated it slowly. 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na f-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (drssociation constant ofthe enzyme ATP complex = 2.5 pM) as well as low concentrations of K'. CrATP was a competitive inhibitor of (Na' + K+)-ATPase.It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na' + K + ) -ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent protein kinase reaction as the phosphate acceptor site. magnetic resonance studies indicate the presence of one divalent metal ion bound at the active site [lo], where the divalent cation seems to maintain the phosphointermediate in the reactive state [ll]. There is evidence that the phosphointermediate formed in the presence of Na+ is converted from an ADPsensitive state into a K t -sensitive state [l, 3,11,12]. The mechanism for this interconversion is unclear, however; the earlier assumption that free Mg2 + might be necessary [1,3,12] has been doubted [8,13,14] since the proposed cyclical changes in the affinity of Mg2' for binding to and release from the enzyme need affinity changes of at least four orders of magni-

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Section: Enzyme and Assaysmentioning

confidence: 99%

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

1980

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“…In view of the variability in potency of other batches of strophanthidin it seemed worth testing their ability to inhibit an Na-K-ATPase preparation. Dose-response curves were constructed for six batches of strophanthidin and one of n-acetyl-strophanthidin (Lilly Ltd) using enzyme isolated from pig brain according to the method of Klodos, Ottolenghi & Boldyrev (1975). Na-K activated Mg2+-dependent ATPase activity was measured as reported by Atkinson, Gatenby & Lowe (1973).…”

Section: Introductionmentioning

confidence: 99%

The function of the sodium pump during differentiation of amphibian embryonic neurones.

Messenger¹,

Warner²

1979

The Journal of Physiology

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SUMMARY1. A method has been developed for studying the differentiation in tissue culture of ectoderm and mesoderm derivatives, dissected from amphibian embryos which have just completed neurulation.2. Neurones, striated muscle cells and pigment cells, together with other unidentifiable cell types, differentiated as a monolayer with approximately the same time course as in the whole embryo. The proportion of different cell types in the cultures was measured quantitatively by cell counting. 3. Treatment of embryos during neurulation with the cardiac glycoside strophanthidin reduced the number of neurones which subsequently differentiated in culture. Other cell types were not affected.4. The relationship between inhibition of neural differentiation and strophanthidin concentration was sigmoid, with maximum inhibition at 10-5 M-strophanthidin and the mid-point at 5 x 10-7 M-strophanthidin. 35 % of neurones differentiating in culture were not affected by glycoside treatment.5. The glycoside hexahydroscillaren A had no effect on neural differentiation. 6. Increasing extracellular potassium to 100 mm during strophanthidin treatment completely protected differentiating neurones from the inhibitory effect of strophanthidin.7. Treatment of embryos with 100 mM-KCl during neurulation had no effect on the subsequent differentiation of neurones.8. Treatment of cultures with an antibody to mouse salivary gland Nerve GrowthFactor reduced the number of neurones by 30 %.9. Exposure to strophanthidin while the embryo moved from the early neural fold stage to the late neural fold stage was as effective in reducing subsequent neural differentiation as treatment throughout neurulation.10. The proportion of nerve cells in the cultures was not affected if strophanthidin treatment ended before the early neural fold stage or did not begin until the late neural fold stage.11. Embryos treated with strophanthidin during neurulation and then allowed to grow into tadpoles developed abnormal nervous systems. 10-6 M-strophanthidin had little effect on the volume of grey matter, but reduced the white matter by 50%.

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“…enzyme activity was determined from the increase in inorganic phosphate under optimal conditions [15]. The specific activity of bovine brain and canine kidney Na,K-ATPase was 120-150 and 1500 ~mol P/ mg/h (37~ respectively.…”

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confidence: 99%

“…The intensity of FR formation was evaluated from the rate of oxidation of 40 p.M ascorbate by measuring absorbance at 265 nm. Na,K-ATPase was preincubated at 4~ with various concentrations of the studied inhibitors, the reaction was terminated by diluting the sample with a buffer-salt solution, and the enzyme activity was measured in medium containing 130 mM NaCI, 20 mM KCI, 3 mM MgCI 2, 30 mM ATP, and 30 mM Pipes (pH 7.4 at 20~ as described elsewhere [15]. The inhibitory control (mixture of aqueous solutions of ascorbate and SN without exposure to light); 2) oxidation of ascorbate in the presence of SN during exposure to light (a mixture of aqueous solutions was exposed to light as described under Materials and Methods, and light absorbance was measured every 5 rain); 3) control (ascorbate solution exposed to light); 4) control (SN solution exposed to light).…”

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confidence: 99%

Is Na,K-ATPase the target of oxidative stress?

1996

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The susceptibility of Na,K-ATPase from bovine brain to various compounds containing active oxygen radicals is assessed. Sodium nitroprusside slightly inhibits Na,K-ATPase, while light-induced NO" radicals (controlled by the rate of ascorbate oxidation) have no effect on the enzyme. When added in concentrations equally effective in the ascorbate oxidation assay, hydrogen peroxide and sodium hypochlorite inhibit Na,K-ATPase by 70 and 25-30%, respectively. The Fe-dinitrosyl-cysteine complex is the most potent (Ko5=20 pM) inhibitor of Na,K-ATPase. It is demonstrated that different free oxygen radicals accumulated in the ischemic brain cause different kinds of damage to Na,K-ATPase.

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Large-scale preparation of Na,K-ATPase from ox brain

Cited by 74 publications

References 11 publications

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

The function of the sodium pump during differentiation of amphibian embryonic neurones.

Is Na,K-ATPase the target of oxidative stress?

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