Figure 1.

Identification of the modification site on rhodopsin by Alexa594–maleimide. Washed ROS, containing mostly rhodopsin, were modified with increasing amounts of Alexa594 (samples 1–3; see Materials and Methods). Next, rhodopsin was digested by thermolysin and subjected to SDS–PAGE (samples 1–3 are lanes 1–3, respectively). The thermolytic F1 fragment contains Cys¹⁴⁰, whereas the F2 fragment contains Cys³¹⁶. Note that at lower concentrations of Alexa594–maleimide, only the F2 fragment was modified. (A) Model of rhodopsin showing the most likely localization of Cys residues and cleavage sites by thermolysin (Th). Cys³¹⁶ is the primary site of modification by Alexa594 (the structure shown in the panel). The model is based on the crystal structure of bovine rhodopsin (5). (B) Proteins on SDS–PAGE gel were visualized with Coomassie Brilliant Blue. Note that the amount of rhodopsin is different among samples. (C) The fluorescent bands were visualized under UV illumination. The level of incorporation of the fluorescent probe into other bands than rhodopsin fragments was less than 4% throughout remaining regions of gel. The amounts of the samples loaded per lane were chosen to contain roughly the same amount of the fluorophore.