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 beta gamma-complex (T beta gamma). 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 beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. 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 beta gamma, and when EDC-treated T beta gamma was incubated with T alpha.
Kosoy
Ana
A
Departamento de Química, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Baruta, Caracas 1081-A, Venezuela.
Möller
Carolina
C
Perdomo
Deisy
D
Bubis
José
J
D000818:Animals; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012243:Rhodopsin / Q000187:drug effects*; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000187:drug effects*; D015398:Signal Transduction; D013194:Staining and Labeling; D015293:Transducin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D006165:Guanylyl Imidodiphosphate; D004024:Dicyclohexylcarbodiimide; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D012243:Rhodopsin; D015293:Transducin; D005022:Ethyldimethylaminopropyl Carbodiimide