Toei et al. 10.1073/pnas.0706914105.
Fig. 5. Characterization of proteoliposomes containing the V-ATPase. (a) Electron microscopic image of liposomes prepared by freeze-thaw sonication method. Liposomes containing the V-ATPase were prepared by freeze-thaw sonication as described in Materials and Methods. The liposomes were stained by uranyl acetate. (b) Histogram of single liposome diameters prepared by freeze-thaw sonication. The diameter of each liposome was directly estimated from EM image of liposomes. The histogram was fitted with Gaussian distribution. The average of diameter of liposomes was estimated to be »122 nm (n = 221). (c) Effect of protein-lipid ratio on ATP synthesis activity. Protein-liposome ratio was ranging from 0.15 to 0.59. The ATP synthesis activity at protein-liposome ratio of 0.29 was represented as 100 % activity. Preparation of proteoliposomes and measurement of ATP synthesis were described in Material and Methods.
Fig. 6. Quality of the 2D crystals. IQ-plot of a typical image included into the merged projection density map. The plot represents the data derived from the Fourier transform of an image after correction of distortions. The IQ (1), proportional to the inverse of the signal-to-noise ratio, is categorized into nine ranges with IQ = 1 as the strongest. Each spot is represented by a square, with the width proportional to its IQ. Spots with IQ values of 1-4 are numbered. The unit cell vectors (a* and b*) of the reciprocal lattice are shown by broken lines. Concentric rings correspond to 1/20, 1/7, 1/4 and 1/3 Ã…-1 (a).
1. Henderson R, Baldwin JM, Downing KH, Lepault J, Zemlin F (1986) Ultramicroscopy 19:147-178.
Table 3. ATP synthesis rate of the V-ATPase dependent on the acidified conditions | |||
 | ATP synthesis rate (s-1) | ||
Acid | DY = 0 mV | DY = 59 mV | DY = 118 mV |
100 mM maleinate | 27 ± 4 | 46 ± 7 | 71 ± 7 |
100 mM malonate | 26 ± 6 | 53 ± 8 | 85 ± 17 |
100 mM succinate | 23 ± 5 | 37 ± 7 | 65 ± 13 |
20 mM maleinate | 11 ± 3 | 21 ± 8 | 33 ± 6 |
100 mM MES | 20 ± 5 | 30 ± 10 | 55 ± 6 |
After soaking with the indicated acidification buffer (pH 2.6-4.9) for 3 min, the acidified proteoliposomes were injected into the base buffer (pH 8.34 ± 0.04) to start the ATP synthesis reaction. Average DpH was 3.28 ± 0.04. The data are the average of three to seven measurements. The error limits are the standard deviations. Details are described in Materials and Methods. | |||
Table 4. Results of the regression analysis for ΔpHeq | ||||||
Condition | Range of ΔpH | Total data number | Correlation coefficient (r) | ΔpHeq | Standard error | 95% confidence interval |
A | 1.55-1.75 | 15 | 0.496 | 1.61 | 0.04 | 1.52-1.69 |
B | 1.80-2.00 | 19 | 0.765 | 1.81 | 0.02 | 1.77-1.85 |
C | 2.00-2.25 | 25 | 0.693 | 2.04 | 0.03 | 1.98-2.09 |
D | 2.25-2.50 | 17 | 0.679 | 2.26 | 0.04 | 2.17-2.34 |
Each ΔpHeq and the standard error values were estimated by nonlinear regression (nls regression) using the program R (www.r-project.org/). | ||||||
Table 5. Results of the regression analysis for n and DGo | |
Total data number | 4 |
The correlation coefficient (r) | 0.999 |
n | 4.00 |
Standard error of n | 0.08 |
95% confidence interval of n | 3.7-4.3 |
DGo (kJ/mol) | 38.9 |
Standard error of DGo | 1.0 |
95% confidence interval of DGo | 35-42 |
The linear regression analysis was used for estimation of n value and DGo. | |