Aluminum fluoride activation of bovine transducin induces two distinct conformational changes in the alpha subunit.
We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan fluorescence, with the rate for the dissociation of holotransducin into its component subunits. Specifically, a beta gamma T complex that was labeled with 5-(iodoacetamido)fluorescein (IAF-beta gamma T) served as a donor for resonance energy transfer and an alpha T-GDP species labeled with eosin 5-isothiocyanate (EITC-alpha TGDP) served as the acceptor. The quenching of IAF-beta gamma T fluorescence emission by the addition of the EITC-alpha TGDP species, due to resonance energy transfer between the IAF and EITC moieities, ranged from 10% to 15%. The association of the transducin subunits was rapid (i.e., within the time period of mixing) and dose-dependent, yielding an apparent Kd of approximately 150 nM for the alpha TGDP/beta gamma T interaction. Unexpectedly, we find that the dissociation of IAF-beta gamma T from an aluminum fluoride-activated alpha TGDP/IAF-beta gamma T complex occurs prior to the onset of the intrinsic fluorescence changes in alpha T that accompany activation of this subunit. Thus, there are at least two structural changes in alpha T that result from the occupation of the gamma-phosphate position in the nucleotide binding cleft of alpha T by aluminum fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)
Mittal
R
R
Department of Pharmacology, Cornell University, Ithaca, New York 14853-6401.
Cerione
R A
RA
Erickson
J W
JW
D017607:Aluminum Compounds / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D005459:Fluorides / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*
D017607:Aluminum Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D006153:Guanosine Diphosphate; C012957:eosine-5-isothiocyanate; C015296:5-iodoacetamidofluorescein; D015293:Transducin; D005459:Fluorides; D004801:Eosine Yellowish-(YS); C032311:aluminum fluoride