@article{Lupfert2001limit-7493,
  year={2001},
  doi={10.1016/S0006-3495(01)75856-0},
  title={Rate limitation of the Na+,K+-ATPase pump cycle},
  number={4},
  volume={81},
  issn={0006-3495},
  journal={Biophysical Journal},
  pages={2069--2081},
  author={Lüpfert, Christian and Grell, Ernst and Pintschovius, Verena and Apell, Hans-Jürgen and Cornelius, Flemming and Clarke, Ronald J.}
}

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    <dcterms:abstract xml:lang="deu">The kinetics of Na+-dependent phosphorylation of the Na+,K+-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na+], and [Mg2+], pH 7.4, and 24°C). The well-established effect of buffer composition on the E2-E1 equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E2) yielded a 1/t value of 59 s1. Addition of MgCl2 (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/ to values of 129, 167, and 143 s1, respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E2 state to an E1 or E1-like state. The results thus demonstrate conclusively that the E2 -&gt; E1 transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E2 -&gt; E1 transition under physiological conditions could be estimated to be ~65 s1 for pig kidney enzyme and 90 s1 for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (~48 s1 and ~43 s1 for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the 1 isoform the E2 -&gt; E1 conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.</dcterms:abstract>
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    <dcterms:bibliographicCitation>First publ. in: Biophysical Journal 81 (2001), pp. 2069-2081</dcterms:bibliographicCitation>
    <dc:contributor>Grell, Ernst</dc:contributor>
    <dc:contributor>Apell, Hans-Jürgen</dc:contributor>
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    <dc:contributor>Cornelius, Flemming</dc:contributor>
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    <dcterms:title>Rate limitation of the Na+,K+-ATPase pump cycle</dcterms:title>
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    <dc:contributor>Pintschovius, Verena</dc:contributor>
    <dc:contributor>Clarke, Ronald J.</dc:contributor>
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