Chemical modification of transducin with iodoacetic acid: transducin-alpha carboxymethylated at Cys(347) allows transducin binding to Light-activated rhodopsin but prevents its release in the presence of GTP.
Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [(3)H]IAA was incorporated per mole of T. Cys(347), located on the alpha-subunit of T (T(alpha)), was identified as the major labeled residue in the [(3)H]IAA-modified holoenzyme. In contrast, Cys(135) and Cys(347) were modified with [(3)H]IAA in the isolated T(alpha). IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233-242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be "locked" in the GDP-bound state of T, even in the presence of R*.
Bubis
J
J
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas 1081-A, Venezuela. jbubis@usb.ve
Ortiz
J O
JO
Möller
C
C
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D006147:Guanine / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D019807:Iodoacetic Acid / Q000737:chemistry*; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors; D015293:Transducin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
D009711:Nucleotides; D010455:Peptides; D013861:Thiocyanates; D006153:Guanosine Diphosphate; D006147:Guanine; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D014357:Trypsin; D015293:Transducin; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid; D019807:Iodoacetic Acid