Rhodopsin-stimulated activation-deactivation cycle of transducin: Kinetics of the intrinsic fluorescence response of the alpha subunit

Journal Article · Tue Jul 31 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00482a003· OSTI ID:6171935

Guy, P M; Koland, J G; Cerione, R A ^[1]

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1. Cornell Univ., Ithaca, NY (USA)

The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on (rhodopsin), while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high (rhodopsin), the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of (32P)Pi production due to (gamma-32P)GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin.

• OSTI ID: 6171935
• Journal Information: Biochemistry; (USA), Vol. 29:30; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
NUCLEOTIDES
BIOCHEMICAL REACTION KINETICS
PROTEINS
FLUORESCENCE
RHODOPSIN
CATTLE
GUANOSINE
LIPOSOMES
PHOSPHORUS 32
TRACER TECHNIQUES
ANIMALS
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LUMINESCENCE
MAMMALS
NUCLEI
NUCLEOSIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHOSPHORUS ISOTOPES
PIGMENTS
PURINES
RADIOISOTOPES
REACTION KINETICS
RIBOSIDES
RUMINANTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques