Chemical modification of transducin with dansyl chloride hinders its binding to light-activated rhodopsin.
Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.
Kosoy
Ana
A
Departamento de Química, Universidad Simón Bolívar, Caracas 1081-A, Venezuela.
Möller
Carolina
C
Perdomo
Deisy
D
Bubis
José
J
D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds / Q000494:pharmacology*; D005123:Eye / Q000033:anatomy & histology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D008027:Light; D008239:Lysine / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013194:Staining and Labeling; D015293:Transducin / Q000737:chemistry / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D015293:Transducin; D008239:Lysine; C029038:dansyl chloride