Characterization of transducin from bovine retinal rod outer segments: mechanism and effects of cholera toxin-catalyzed adp-ribosylation
Transducin, a guanine nucleotide-binding protein consisting of two subunits (T/sub ..cap alpha../ and T/sub ..beta gamma../), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T/sub ..cap alpha../ subunit is an activator of the phosphodiesterase, and the function of the T/sub ..beta gamma../ subunit is to physically link T/sub ..cap alpha../ with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T/sub ..cap alpha../ has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T/sub ..cap alpha../ with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(..beta..,..gamma..-im ido)triphosphate (Gpp(NH)p) and T/sub ..beta gamma../ were present at concentrations equal to that of T/sub ..cap alpha../ and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T/sub ..cap alpha../xGpp(NH)p, T/sub ..beta gamma../, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T/sub ..cap alpha../ coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 x 10/sup -3//s, which was 22-fold slower than the rate for the unmodified transducin. 30 references, 9 figures, 1 table.
- Research Organization:
- Univ. of Rochester Medical Center, NY
- OSTI ID:
- 6109377
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 259:10
- Country of Publication:
- United States
- Language:
- English
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PHOSPHODIESTERASES
PROTEOLYSIS
RETINA
RHODOPSIN
RIBOSE
TOXINS
ALDEHYDES
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ANTIGENS
BACTERIAL DISEASES
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CARBOHYDRATES
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ENZYMES
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INFECTIOUS DISEASES
INFORMATION
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MAMMALS
MATERIALS
MONOSACCHARIDES
NUCLEOTIDES
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANS
PENTOSES
PIGMENTS
REACTION KINETICS
RUMINANTS
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SENSE ORGANS
TOXIC MATERIALS
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520200* - Environment
Aquatic- Chemicals Monitoring & Transport- (-1989)