Identification of functionally important acidic residues in transducin by group-specific labeling.

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T-alpha) and the betagamma-complex (T-betagamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T-alpha was combined with DCCD-treated T-betagamma. However, the binding of guanine nucleotides to the reconstituted T was similar to50% inhibited when DCCD-labeled Ta was incubated with T-betagamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T-alpha was combined with T-betagamma, and when EDC-treated T-betagamma was incubated with T-alpha.