Figure 5. Michaelis-Menten Kinetics.

Kinase reactions were carried out with rhodopsin (2.5–20 µM), 100 nM WT GRK2, GRK2-Y281A, GRK2-P473E, and GRK2-I485A or 400 nM GRK2-V477D, and 1 mM [γ-³²P] ATP (~0.2 dpm/fmol) for 2 min in the light. Rhodopsin was separated by electrophoresis, stained, and excised to quantitatively determine phosphate transferred. The data are derived from three experiments carried out in duplicate and error bars represent SEM. Initial rate data was fit to Michaelis-Menten rate equation and values for K_M and k_cat are given in Table 1.