. | Supernatant . | . | Pellet . | . | . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|
Treatment . | MsGC-β3 . | MsGC-β3ΔC338 . | MsGC-β3 . | (%) . | MsGC-β3ΔC338 . | (%) . | ||||
None (50 mmol l-1 Tris-HCl) | 0.98±0.03 | 2.30±0.05 | 5.30±0.30 | 84 | 3.77±0.18 | 62 | ||||
0.5 mol l-1 NaCl | 0.78±0.02 | 1.96±0.03 | 4.52±0.12 | 85 | 4.44±0.24 | 69 | ||||
1% Chaps | 2.16±0.04 | 1.60±0.03 | 6.20±0.10 | 74 | 4.74±0.20 | 75 | ||||
1% Digitonin | 3.21±0.04 | 3.14±0.08 | 6.27±0.15 | 66 | 4.24±0.07 | 67 | ||||
1% Octyl β,D-thioglucopyranoside | 3.80±0.04 | 5.49±0.07 | 7.12±0.44 | 65 | 2.60±0.04 | 32 | ||||
1% Triton X-100 | 4.36±0.03 | 4.29±0.05 | 4.81±0.20 | 52 | 5.30±0.10 | 55 | ||||
1% Tween 20 | 2.19±0.10 | 4.01±0.09 | 6.20±0.10 | 74 | 4.80±0.27 | 54 |
. | Supernatant . | . | Pellet . | . | . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|
Treatment . | MsGC-β3 . | MsGC-β3ΔC338 . | MsGC-β3 . | (%) . | MsGC-β3ΔC338 . | (%) . | ||||
None (50 mmol l-1 Tris-HCl) | 0.98±0.03 | 2.30±0.05 | 5.30±0.30 | 84 | 3.77±0.18 | 62 | ||||
0.5 mol l-1 NaCl | 0.78±0.02 | 1.96±0.03 | 4.52±0.12 | 85 | 4.44±0.24 | 69 | ||||
1% Chaps | 2.16±0.04 | 1.60±0.03 | 6.20±0.10 | 74 | 4.74±0.20 | 75 | ||||
1% Digitonin | 3.21±0.04 | 3.14±0.08 | 6.27±0.15 | 66 | 4.24±0.07 | 67 | ||||
1% Octyl β,D-thioglucopyranoside | 3.80±0.04 | 5.49±0.07 | 7.12±0.44 | 65 | 2.60±0.04 | 32 | ||||
1% Triton X-100 | 4.36±0.03 | 4.29±0.05 | 4.81±0.20 | 52 | 5.30±0.10 | 55 | ||||
1% Tween 20 | 2.19±0.10 | 4.01±0.09 | 6.20±0.10 | 74 | 4.80±0.27 | 54 |
COS-7 cells were transiently transfected with MsGC-β3 or MsGC-β3ΔC338 as described in Materials and methods and homogenized in 50 mmol l-1 Tris HCl, pH 7.5. Salt or detergents were added to separate samples as shown, which were then incubated for 30 min at 4°C and centrifuged at 14 000 g for 30 min at 4°C. The pellet was resuspended in the same detergent that was used for solubilization and both supernatant and pellet fractions assayed for guanylyl cyclase activity.
Values are the means ± S.E.M. of 3 measurements.
% indicates percentage of total enzyme activity in pellet fractions.