Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin.
The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.
Ortiz
J O
JO
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Bubis
J
J
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008027:Light; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D011487:Protein Conformation / Q000187:drug effects; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology; D019163:Reducing Agents / Q000737:chemistry / Q000494:pharmacology; D012243:Rhodopsin / Q000378:metabolism* / Q000494:pharmacology / Q000528:radiation effects; D012374:Rod Cell Outer Segment / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013267:Stilbenes / Q000737:chemistry / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology; D013451:Sulfonic Acids / Q000737:chemistry / Q000494:pharmacology; D013861:Thiocyanates / Q000737:chemistry / Q000494:pharmacology; D015293:Transducin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
D005456:Fluorescent Dyes; D006150:Guanine Nucleotides; D011725:Pyridines; D019163:Reducing Agents; D013267:Stilbenes; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D013451:Sulfonic Acids; D013861:Thiocyanates; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D006165:Guanylyl Imidodiphosphate; D012243:Rhodopsin; D015293:Transducin; C029351:4-vinylpyridine; C008658:2-nitro-5-thiocyanobenzoic acid