Figure 4.

Native PhLP binds CCT. (A) ¹²⁵I-G_tα, ³⁵S-PhLP, and ³⁵S-Pd at a final concentration of 5 μM were denatured in 6 M urea or maintained in their native state in buffer without urea and then diluted 100-fold in the presence of CCT from rabbit reticulocyte lysates. Samples were immunoprecipitated by using the anti-TCP-1α antibody and subjected to SDS/PAGE and phosphorimaging. N and D indicate respectively native or urea-denatured samples. The value of urea-treated G_tα was set at 100% for the ¹²⁵I-labeled samples and the value of urea-treated PhLP was set at 100% for the ³⁵S-labeled samples. Error bars represent the standard deviation from three separate experiments. (B) PhLP was denatured under the same conditions as above. Urea-treated (○) or nontreated (▴) PhLP samples were then incubated with 0.2 μM ¹²⁵I-G_tα, 0.2 μM G_tβγ, and urea-stripped retinal rod outer segment membranes (1.0 μM rhodopsin). The binding of G_t to light-activated rhodopsin was then assayed as described (15). The final dilution of urea had no effect on the total binding. Error bars represent the standard deviation from three separate experiments.