Photoaffinity labeling of a single receptor for alpha-thrombin on mouse embryo cells.

High purity human Lu-thrombin was labeled with “‘Iby lactoperoxidase-catalyzed iodination and was conjugated with the photoreactive reagent N-(4-azido-2nitrophenyl)-2-diaminoethane (NAPEDE) by carbodiimide coupling. Cultured mouse embryo fibroblasts, which respond mitogenically to cu-thrombin, bound the conjugated and unconjugated enzyme similarly, indicating that the NAPEDE group did not significantly alter binding of thrombin to its receptors. Photoactivation of NAPEDE-1261-cu-thrombin bound specifically to cells led to the formation of a lz51-labeled component ofM, = 80,500 + 3,300 (N = 4) in reducing or nonreducing sodium dodecyl sulfate-containing polyacrylamide gels. A smaller component of M,. = 30,250 + 4,000 (N = 4) co-migrated with NAPEDE-‘251-cY-thrombin or NAPEDE-‘2SI-cw-thrombin photoactivated in the absence of cells. The radioactivity in the larger component was proportional to the specific binding of NAPEDE-‘251-cu-thrombin to cells and was not detected when an 80-fold excess of unlabeled a-thrombin was included to inhibit specific binding of NAPEDE-lZ51-athrombin to receptors. Approximately 60% of the NAPEDE-‘251-a-thrombin specifically bound to cells