Supplementary information Fig. S1.

Transcription

1 Supplementary information Kinetic equivalence of transmembrane ph and electrical potential differences in ATP synthesis Naoki Soga, Kazuhiko Kinosita, Jr., Masasuke Yoshida and Toshiharu Suzuki Fig. S1. Dependence of ATP synthesis activity on the incubation time for acidification. 30 µl of proteoliposome suspension was mixed with 70 µl of the acidic buffer of ph 6.2. After indicated times, the initial rate of synthesis, v, was measured at ph out = 8.0, [K + ] out = 158 mm and [K + ] in = 0.5 mm. Two independent reconstitutions are distinguished by colors. Indicated ψ is a nominal value calculated from the Nernst equation.

2 Fig. S2. Removal of contaminant K + from crude lipid. See Materials and methods for details. (A) Reduction of K + level by repeated centrifugation and freezing/thawing cycles. Small circles show individual measurements after a cycle, and large circles their averages. Values at cycle 0 show contamination in the untreated lipid. (B) Determination of K + levels before and after the purification. Small circles, the K + level at the indicated lipid concentration determined by adding known amounts of K + and extrapolating back to no addition; large circles with error bars, average and standard deviations.

3 Fig. S3. The effect of low [K + ] in on the ATP synthesis activity. Values of pmf shown are calculated from the Nernst equation. Colors distinguish different [K + ] in as indicated. (A) Time courses at constant ph of 1.6 (ph out = 8.0). ψ was also kept around nominal 80 mv by changing [K + ] in and [K + ] out simultaneously. Nominal pmf values are shown in mv, and the initial rate of synthesis, v, in s -1. Note the faster deceleration of synthesis rate at lower [K + ] in, which presumably reflects faster decay of ψ. When fluxes of ions other than K + is not negligible, electrochemical equilibrium of K + across the membrane (the basis of the Nernst equation) is not reached and K + continues to flow into the liposome. (B) The rate of synthesis plotted against the nominal ψ calculated from the Nernst equation. Deviations are noticeable at [K + ] in < 1 mm. Vertical gray bar indicates the pmf at which the time courses in A were obtained.

4 Fig. S4. Apparent pmf dependence of all synthesis activities including those at [K + ] in < 1 mm. Low [K + ] in data are added to Figure 4A in the main text. Data at ph of 2.4 (ph in = 5.6) are excluded. Data at [K + ] in < 1 mm are emphasized with larger symbols. (A) Uncorrected pmf dependence. Values of pmf are calculated from the Nernst equation. (B) Corrected pmf dependence. When passage of ions other than K + is not negligible, ψ is approximated by the Goldman equation: ψ = k BT e ln P K +[K + ] out + P i i [M + i ] out + P j j [A j ] in P K +[K + ] in + P i [M + i ] in + P j [A j ] out i j where M + i s are (monovalent) cations other than K +, A j s anions, and P i and P j the permeability of respective ions (1). When the K + terms far exceed others, the Goldman equation reduces to the Nernst equation. At low [K + ] in, other terms in the denominator may not be negligible. Because the concentrations of other ions are essentially constant (we changed [Na + ] in depending on [K + ] in, but at low [K + ] in the change was small), we approximate the Goldman equation above with ψ =(k B T /e)ln([k + ] out /[K + ] in + α) where α is a constant. The numerator must also be corrected at low [K + ] out, but we ignore this because the measured activity was low at low [K + ] out (low ψ): for all activities above 2 s -1, [K + ] out was at least 5 mm. We sought for α that would minimize the spread in panel A. Panel B shows the results, with α = 0.5 mm (if Na + and/or Cl - are the permeant ions, their permeabilities would be more than two orders of magnitude lower than valinomycin assisted K + ). The overlaps obtained by the correction suggest that the apparent anomalies at low [K + ] in are indeed due to the leakage of other ions. The faster decay of ψ at low [K + ] in (Figure S3A) is consistent with this interpretation.

5 Supplementary Reference 1 Goldman D. E. (1943) J. Gen. Physiol. 27, 37-60