1. Electrogenic Partial Reactions of the Gastric H,K-ATPase
Anna Diller, Olga Vagin, George Sachs, Hans-Jürgen Apell 
Biophysical Journal 
Volume 88, Issue 5, Pages (May 2005)
DOI: /biophysj
Copyright © 2005 The Biophysical Society Terms and Conditions

2. Figure 1
Simplified pump cycle of the H,K-ATPase on the basis of the Post-Albers scheme of P-type ATPases. E1 and E2P represent the two basic conformations of the protein. In E1 the ion-binding sites are accessible from the cytoplasmic side (left side of the cycle), in E2P from the luminal side (right side of the cycle). In both occluded states, (H2)E1P and (K2)E2, the ions are unable to exchange with either aqueous phase. The stoichiometry of two H+ and two K+ ions, which are moved in opposite directions, accounts for the overall electroneutrality of the transport process.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

3. Figure 2
Time course of the fluorescence signal of the electrochromic styryl dye RH421 indicating substrate-dependent partial reactions of the H,K-ATPase. The initial state of all four experiments shown was E1 (Fig. 1), which was obtained with 10μg H,K-ATPase in standard buffer at pH 8.5 and with no K+ ions present. This level was defined to be F0. The fluorescence scale on all four panels is the same. (A) Addition of HCl (14.5mM) produced a pH decrease to 6.5, ATP was added to obtain a concentration of 2mM in the cuvette, and KCl was added to obtain a final concentration of 240mM. (B–D) Addition of the substrates in varying order produced different sequences of protein states as indicated in the panels. The label “E2(K2)+?” was chosen to point out that in the presence of 30μM (free) H+ and 2mM ATP the pump has all substrates to cycle, and the reduced fluorescence decrease, when compared to the maximal possible in the state (K2)E2, indicates that other states were also populated. Although most responses upon substrate addition were fast, i.e., within stirring time in the cuvette, binding of H+ to the luminal binding sites showed a slow kinetics with time constants τ>10s (B).
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

4. Figure 3
Titration of the cytoplasmic binding sites of the H,K-ATPase with H+ and K+ ions as detected by the RH421 fluorescence. The mutual effect of both ions on each other indicates competitive behavior in the binding sites. (A) pH titration experiments in the presence of the indicated K+ concentrations. (B) K+ titrations in buffer of various pH as indicated. The lines drawn through the experimental data were obtained by least-square fits of Eq. A12 with the one set of kinetic parameters: KH,1=2.0×10−7 M (pK 6.7), KH,2=3.2×10−5 M (pK 4.5), KK,1=11mM, and KK,2=16mM. Data points are mean values of at least three sets of experiments. The error bars were removed for the sake of clarity.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

5. Figure 4
Titration of the luminal binding sites of the H,K-ATPase with H+ and K+ ions as detected by the RH421 fluorescence. The mutual effect of both ions on each other indicates competitive behavior in the binding sites. (A) pH titration experiments in the presence of the indicated K+ concentrations. (B) K+ titrations in buffer of various pH as indicated. The lines drawn through the experimental data were obtained by least-square fits according to Eq. A26 of the reaction model described in Appendix II. The reaction constants are given in Table 1. Data points are mean values of at least three sets of experiments. The error bars were removed for the sake of clarity.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

6. Figure 5
Dependence of the specific enzymatic activity on K+ concentration in the buffer. The activity was measured by Pi determination with malachite green at 37°C. The concentration dependence was fitted by simple binding isotherm with a Km=0.11±0.01mM and EA,max=140.5±3μmol Pi per milligram protein and hour.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

7. Figure 6
Ion selectivity of the binding sites in the two principal conformations, (A) E1 and (B) P-E2 in standard buffer at pH 8.5 for Na+, K+, and NH4+. The fluorescence decrease is proportional to the occupation of the binding sites with cations and could be fitted with a simple binding isotherm (Eq. 2). For details see text.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

8. Figure 7
Expanded version of ion binding and release steps of the pump scheme in Fig. 1. (A) The simplest possible mechanism for the binding sites presented to the cytoplasm is a linear, sequential arrangement that excludes mixed occupation of the ion-binding sites. State K2E1 is shown in parentheses to indicate that it is not explicitly treated in the mathematical treatment due to the fact the steady state of the reaction K2E1 ↔ K2E2 is strongly shifted to the right side. This reaction scheme has the minimum number of kinetic parameters, the four equilibrium dissociation constants, KH,2, KH,1, KK,1, KK,2. (B) In the presence of ATP the pump is able to present the sites alternatively to both aqueous phases. Therefore, the whole cycle of Fig. 1 has to be expanded. The luminal equilibrium dissociation constants were introduced as LH,2, LH,1, LK,1, and LK,2. Since this model was applied on equilibrium titration experiments, all other reaction steps may be comprised in two reaction steps with the rate constants, kA and kP. (Due to the negligible ADP and Pi concentrations in the experimental buffer, back reactions were omitted.) In addition, the rate constant, kD, is included. It indicates the proposed pathway for spontaneous dephosphorylation of the enzyme in the absence of luminal K+.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions