Table S1.
Functional analysis of mutants
| Enzyme | Maximal enzyme turnover rate,* min−1 | K0.5 (Na+),† μM | K0.5 (K+),† μM | K0.5 (VO43−),† μM | K0.5 (ATP),† μM | Inhibition of Na+-ATPase at high Na+‡ |
| Wild type α1 | 8,474 ± 165 (n = 11) | 483 ± 18 (n = 8) | 624 ± 13 (n = 14) | 2.5 ± 0.1 (n = 23) | 432 ± 25 (n = 9) | ++ |
| C932A | 6,896 ± 354 (n = 6) | 476 ± 29 (n = 6) | 518 ± 19 (n = 3) | 2.5 ± 0.1 (n = 3) | 494 ± 40 (n = 3) | +++ |
| 1.0× | 1.2× ↓ | 1.0× | 1.1× ↑ | |||
| C932F | 7,281 ± 179 (n = 6) | 35,292 ± 3,498 (n = 7) | 996 ± 35 (n = 3) | 5.1 ± 0.2 (n = 5) | 220 ± 75 (n = 4) | − |
| 73.1× ↑ | 1.6× ↑ | 2.1× ↑ | 2.0× ↓ | |||
| C932L | 6,448 ± 383 (n = 6) | 14,339 ± 794 (n = 5) | 459 ± 7 (n = 3) | 3.7 ± 0.2 (n = 3) | 231 ± 26 (n = 3) | − |
| 29.7× ↑ | 1.4× ↓ | 1.5× ↑ | 1.9× ↓ | |||
| C932R | 7,031 ± 258 (n = 6) | 2,440 ± 212 (n = 6) | 1,310 ± 54 (n = 3) | 3.1 ± 0.1 (n = 5) | 189 ± 10 (n = 4) | + |
| 5.1× ↑ | 2.1× ↑ | 1.2× ↑ | 2.3× ↓ |
SEs and number of independent determinations (n) are indicated. Fold change (×) relative to wild type is indicated by number with arrow.
Data illustrated in Fig. 3A. The Na+,K+-ATPase activity was determined at 37 °C in the presence of 30 mM histidine buffer (pH 7.4), 130 mM NaCl, 3 mM ATP, 3 mM MgCl2, 1 mM EGTA, ouabain to inhibit the endogenous enzyme, and 20 mM KCl. The enzyme turnover rate was calculated as the ratio between the Na+,K+-ATPase activity and the active site concentration (maximum phosphorylation from [γ-32P]ATP measured at 0 °C in the presence of 150 mM NaCl and oligomycin).
Extracted from the data in Fig. 3 B and D and Fig. S2 A and B by nonlinear regression fitting procedures (equations are shown in SI Methods). The best fit shown as a line in the figures.
Semiquantitative interpretation of the data in Fig. 3C (inhibition at high Na+ indicated by +, and lack of inhibition indicated by −).