Figure 4.

Melanopsin stimulates GTP-γ-³⁵S uptake by transducin. (A) Melanopsin-mediated GTP-γ-³⁵S binding in bright white light (□) and in the dark (■). Reaction rates were 0.04 pmol GTP-γ-³⁵S bound/s/pmol receptor for the light reaction and 0.02 pmol GTP-γ-³⁵S bound/s/pmol receptor for the dark reaction. In this assay, reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S, and 9.14 nM melanopsin in a buffer containing 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM MgCl₂, and 1 mM DTT. Circles represent GTP-γ-³⁵S binding by untransfected COS-1 membranes in bright white light (○), and in the dark (●). Reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S and untransfected COS-1 membranes in the Tris buffer described above. (B) Melanopsin is activated most efficiently by blue light. Mean rates of melanopsin-catalyzed GTP-γ-³⁵S binding following illumination with UV (381 ±10 nm), blue (443 ±19 nm), green (516 ±30 nm), and red (> 700 nm) light. Illumination was provided by a calibrated light source. The samples were illuminated for 10 s. and the rate of transducin activation was determined. The irradiance (photons/s/cm²) was: UV—1.3 ×10¹³; Blue—1.54 ×10¹³; Green—2.72 ×10¹³; Red—1.9 ×10¹⁵. Wavelengths were selected using bandpass or longpass filters. Intensities were chosen to provide clear discrimination amongst action spectra for candidate pigments that have absorption maxima in the spectral range from the UV to the green. The intensity of red light was 100-fold greater than the other stimuli to rule out any contribution of photoactivation by light outside the intended spectral bands. The GTP-γ-³⁵S binding rate in the dark (4.8 ×10⁻⁴ pmol/s) was subtracted from raw rate values to generate the data shown (Mean ± SD). Reaction mixtures contained 2.1 μM transducin, 3 μM GTP-γ-³⁵S, and 0.3 nM melanopsin.