Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.

Nørby, Jens G.; Klodos, Irena; Christiansen, Niels Obel

doi:10.1085/jgp.82.6.725

Cited by 105 publications

(33 citation statements)

References 51 publications

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“…One way of explaining the discrepancy between the two estimates of k2 obtained from Mardh's data would be to suppose that, with his preparation and in the conditions he used, k2 was much increased by the presence of potassium. A large effect of potassium on k2 has been thought unlikely by N0rby, Klodos & Christiansen (1984) because, in various experiments designed to study the effects of potassium on the disappearance of pre-formed phosphoenzyme, the slow phase of the biphasic curve that describes the disappearance of phosphoenzyme following the addition of potassium was not significantly faster than the rate of disappearance of phosphoenzyme in the absence of potassium (Tonomura & Fukushima, 1974;Kuriki & Racker, 1976;Hara & Nakao, 1981). In MArdh's experiments, however, the slow phase of disappearance of pre-formed phosphoenzyme after the addition of potassium had a rate constant of about 43 s-1 (see Fig.…”

Section: Compatibility Of Rate Constants and Observed Fluxesmentioning

confidence: 99%

The occlusion of sodium ions within the mammalian sodium‐potassium pump: its role in sodium transport.

Glynn¹,

Hara²,

Richards³

1984

The Journal of Physiology

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The hypothesis that the ADP-sensitive form of phosphorylated Na+, K+-ATPase contains occluded sodium ions has been tested by a procedure which involves (i) modifying the enzyme with alpha-chymotrypsin or N-ethylmaleimide (NEM) so that the ADP-sensitive form is more stable than it is in the native enzyme, (ii) phosphorylating the modified enzyme with ATP in the presence of labelled sodium ions, and (iii) forcing the phosphorylated enzyme rapidly through a cation-exchange column and measuring the labelled sodium in the effluent. The results show that ADP-sensitive phosphoenzyme prepared from alpha-chymotrypsin- or NEM-modified Na+, K+-ATPase is able to carry labelled sodium ions through a cation-exchange resin. This behaviour was not seen with native Na+, K+-ATPase or when phosphorylation was prevented by the omission of magnesium ions or by the substitution of adenylyl(beta, gamma-methylene)diphosphonate (AMP-PCP) for ATP. The occluded sodium ions were rapidly released when the phosphoenzyme was dephosphorylated by ADP. When alpha-chymotrypsin-modified enzyme was phosphorylated by ATP with 1 mM-sodium in the medium, close to three sodium ions were occluded per phospho group. The stoicheiometry at much lower sodium concentrations could not be determined satisfactorily. A consideration of the rate constants of the reactions thought to be involved in the occlusion of sodium and in the release of sodium from the occluded state shows that, so far as they are known, these constants are compatible with the hypothesis that the occluded-sodium form of the phosphoenzyme plays a central role in sodium transport through the pump.

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Section: Compatibility Of Rate Constants and Observed Fluxesmentioning

confidence: 99%

The occlusion of sodium ions within the mammalian sodium‐potassium pump: its role in sodium transport.

Glynn¹,

Hara²,

Richards³

1984

The Journal of Physiology

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“…Steady-state EP includes at least three different intermediates (14). We next looked at the ligand-dependent dephosphorylation of the control enzyme, and the enzyme totally lacked its K ϩ -dependent activity (treated with 100 M CPZ).…”

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confidence: 99%

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Mahmmoud

2008

Proc. Natl. Acad. Sci. U.S.A.

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Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K ؉ . Studies with purified membranes revealed that CPZ reduced Na ؉ -dependent phosphorylation by interference with Na ؉ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K ؉ . Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive 22 Na ؉ influx into K ؉ -loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na ؉ cycle from an Na ؉ /K ؉ cycle in the pump. The Na ؉ cycle possibly involves transport through the recently characterized Na ؉ -specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na ؉ efflux by modifying the transport stoichiometry of single pump units.sodium pump ͉ uncoupling ͉ potassium occlusion N a,K-ATPase (NKA), a P-type cation pump (1), utilizes energy from ATP hydrolysis to exchange intracellular Na ϩ for extracellular K ϩ against their electrochemical potential gradients. The functional pump contains an ␣-subunit that undergoes substrateinduced conformational transitions, coupling ATP hydrolysis to ion transport, and a ␤-subunit important for maturation and targeting of the complex to the membrane (2).According to the Albers-Post scheme, occlusion of three intracellular Na ϩ ions promotes phosphorylation of the ␣-subunit from ATP, whereas occlusion of two extracellular K ϩ ions promotes dephosphorylation (2). Depending on the specific ionic composition on both sides of the membrane, the pump catalyzes several partial reactions (3). In the absence of K ϩ , NKA mediates ATP-driven Na ϩ transport processes, including exchange as well as uncoupled efflux (reviewed in ref. 4). Furthermore, in inside-out vesicles of human red cells, the ratio of net 22 Na ϩ influx/ 86 Rb ϩ efflux was reduced at sub-millimolar cytoplasmic Na ϩ concentrations. This finding indicates that under this particular condition, the pump can function with less than a maximum number of sites filled with Na ϩ ions (5).In sarcoplasmic reticulum (SR) Ca 2ϩ -ATPase (SERCA), the best characterized P-type pump, ATP hydrolysis can take place without Ca 2ϩ transport to allow for thermogenesis (6). This uncoupling is prominent in the presence of a Ca 2ϩ gradient across the SR membrane. Recent studies have shown that sarcolipin, a small SERCA regulator, increases heat production by this pump (7). This finding demonstrates a regulatory inter...

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“…The existence of an external K-stimulated ouabain-sensitive ATP-ADP exchange resulting from an increased rate of ATPase activity has important implications for the mechanisms involved in the whole reaction cycles. In opposition to the view that the early steps of the ATP hydrolysis are the same in the presence of Na and of Na + K, and lead to the formation of E1P and E2P intermediates (for references see Glynn & Karlish, 1975;Robinson & Flashner, 1979;Jorgensen, 1982;De Weer, 1983;Beauge, 1984), it has been proposed that Na-ATPase and Na,K-ATPase activates go through different cycles; this hypothesis considers that with Na + K in the media E1P and E2P are not formed (Plesner, Plesner, Norby & Klodos, 1981;Norby, Klodos & Christiansen, 1983). The fact that external K stimulates both ouabain-sensitive ATP hydrolysis and ATP-ADP exchange appears extremely difficult to explain unless the cycles of ATP hydrolysis give rise to the same intermediates in the presence of Na alone and of (Na + K); therefore this interpretation excludes different cycles for Na and Na + K-ATPase activities.…”

Section: Discussionmentioning

confidence: 99%

Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K‐ATPase.

Beaugé¹,

Campos²

1986

The Journal of Physiology

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SUMMARY1. Interactions ofNa, K, Ca and Mg ions with partially purified Na,K-ATPase from pig kidneys were investigated using as a tool simultaneous determinations of adenosine 5'-triphosphate (ATP)-adenosine 5'-diphosphate (ADP) exchange and ATPase activity catalysed by the enzyme.2. In the presence of 120 mM-NaCl and 0 5 mM-MgCl2, the effects 12 mM-KCl on ATP-ADP exchange depended on the ATP and ADP concentrations: stimulation (about 10-fold) with 3 mM-ATP-075 mM-ADP, no effect with 0-04 mM-ATP-0 01 mM-ADP and inhibition when ATP and ADP concentrations were 0 003 mm and 0-001 mm respectively. The apparent affinities (KO.5) for K stimulation of ATP-ADP exchange and Na,K-ATPase activity were indistinguishable from each other both at 20 mmand 120 mM-Na. All Na and Na + K-dependent exchanges were completely abolished by 10-4 M-ouabain.3. In the absence of K, intermediate Na concentrations were inhibitory of ouabain-sensitive ATP-ADP exchange and ATPase activity. This Na inhibition was more pronounced at low than at high ionic strength and was more noticeable in the exchange reaction. K ions antagonized these Na effects; this K-Na antagonism was more effective on ATPase than on the ATP-ADP exchange.4. Ca ions, in the absence of Mg, could sustain ATP-ADP exchange. Qualitatively the behaviour of the exchange reaction was similar to that observed with Mg. Quantitatively the rates were always lower with Ca. In the presence ofMg, at low ionic strength and at concentrations which stimulated phosphatase activity, Ca ions were not able to counteract Na inhibition of ATPase and ATP-ADP exchange or to stimulate ATPase activity in the presence of Na. This is another indication of the different reactivity of the K sites involved in phosphatase and ATPase activities as well as of those responsible for the release of Na inhibition of ATPase and exchange reaction.5. In K-free solutions containing 0-5 mM-Na and low ionic strength, Mg was a powerful inhibitor of ouabain-sensitive ATPase and ATP-ADP exchange; the curves relating remaining activities to Mg concentrations were identical for both reactions. When Na was increased to 5 mM, ATP-ADP exchange became more sensitive and ATPase more resistant to Mg inhibition. These data are consistent with (i) a Na-Mg antagonism at the phosphorylation step, and (ii) a Na-Mg synergism in blocking the 1 PHY 375ATP-ADP EXCHANGE AND Na,K-ATPase E2PNa-E1PNa transformation. At high ionic strength, with higher concentrations of Na alone and of Na + K, Mg was also a strong inhibitor of ATP-ADP exchange; in this case the possible mechanism is less clear, although K ions could, to some extent, counteract this Mg effect. 6. The present results are consistent with Na inhibition of ATP-ADP exchange and ATPase activity by stabilizing the E2P conformation. This stabilization is selective in that it is easier to overcome in the forward direction (towards ATP hydrolysis) than in the backward directions (leading to ATP synthesis). These results seem to be best explained on the basis that K stimulation of ouabain...

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Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.

Cited by 105 publications

References 51 publications

The occlusion of sodium ions within the mammalian sodium‐potassium pump: its role in sodium transport.

The occlusion of sodium ions within the mammalian sodium‐potassium pump: its role in sodium transport.

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Effects of mono and divalent cations on total and partial reactions catalysed by pig kidney Na,K‐ATPase.

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