Table 1.
Adenylyl cyclase stimulation by αS-Q170A [pmol cAMP/ml translation product/15 min (percent of WT)]
| αS | GTP, 100 μM | Isoproterenol, 10 μM, + GTP, 100 μM | GTPγS, 100 μM | AlF4−b* |
|---|---|---|---|---|
| WT | 25 ± 10 | 233 ± 5 | 259 ± 21 | 225 ± 12 |
| Q170A | 29 ± 6 | 246 ± 24 (106 ± 11) | 305 ± 4 (118 ± 10) | 113 ± 9 (50 ± 5) |
| R258A† | (24 ± 3) | (132 ± 6) | (62 ± 9) |
In vitro transcription/translation products were mixed with purified cyc- membranes and were assayed for adenylyl cyclase stimulation at 30°C. Results are expressed as the mean ± SD, (σn−1) of triplicate determinations and are representative of three independent experiments. The levels of expression of αS-WT and -Q170A were similar as determined by in vitro translation with [35S]methionine, SDS/PAGE, and phosphorimaging. Background values determined from mock transcription/translation reactions (in pmol cAMP/ml translation medium/15 min; GTP, 39 ± 4; isoproterenol/GTP, 46 ± 1; GTPγS, 58 ± 2; and AlF4−; 68 ± 8) were subtracted from each determination.
10 mM NaF, 10 μM AlCl3, and 100 μM GDP.
The results for αS-R258A (shown here as percent of αS-WT) have been published (18) and are shown here for comparison with αS-Q170A.