Figure 3.

Purification of α₃^chiγ and ATP hydrolytic activity of Gsα₃β₃γ and α₃^chiγ. A, the chromatogram shows an elution profile of α₃^chiβ₃γ using a Resource-Q column (6 ml). The inset in the figure reveals a SDS gel, which corresponds to the shaded area (gray) of the elution peak. The eluted α₃^chiβ₃γ fractions were pooled and applied on the gel. Lanes 1 and 2 reveal the purified α₃^chiβ₃γ and protein markers, respectively. The protein size in kDa is indicated on the right. B, continuous ATPase activity of Gsα₃β₃γ and α₃^chiβ₃γ measured at 2 mm MgATP, 37 °C. Decrease in NADH absorption at 340 nm is plotted against time as dotted lines. The black lines show the linear least square fit for the first 10 s. C, specific ATPase activities of Gsα₃β₃γ and α₃^chiβ₃γ determined from the slope of the least square fit. Values are the mean of 10 determinations. D, ATPase profile of nucleotide-depleted Gsα₃β₃γ (—) in a continuous ATPase activity assay measured at 2 mm MgATP. Bound MgADP was removed prior to activity measurements by incubating the protein with 5 mm Na-P and 10 mm EDTA for 30 min at 4 °C, and passing the enzyme samples through a Superdex 300^TM column (GE Healthcare). (····) ATPase profile of Gsα₃β₃γ, which was preincubated with 100 μm MgADP for 30 min, revealing the MgADP inhibition effect, and confirming that tightly bound MgADP was removed due to the nucleotide depletion process of nucleotide-depleted Gsα₃β₃γ (—) described above. E, specific ATPase activities of the nucleotide-depleted Gsα₃β₃γ and α₃^chiβ₃γ. Values are the mean of three determinations.