The nucleotide binding site of the GTP-binding protein (transducin) in bovine rod outer segments

AbstractThe role of 48-kDa protein in Visual transduction remains unresolved. Two hypotheses for its role in quenching the light activation of cyclic GMP cascade suggest that the protein binds to either phosphodiesterase or phosphorylated rhodopsin. Since the protein is also reported to bind ATP, we anticipated that the protein may have ATP hydrolyzing activity, and in analogy with the GTP-binding protein of the rod outer segments, such activity may be greatly enhanced by the elements of transduction cyclic GMP cascade, permitting the protein to function cyclically as GTP-binding protein does. We found that purified 48-kDa protein hydrolyzes ATP but at a slow rate of 0.04–0.05 per min. The Km for ATP is about 45–65 μM. The activity is inhibited noncompetitively by ADP with a Ki of about 50 μM. The ATPase activity of 48-kDa protein is not affected by rhodopsin, bleached rhodopsin, phosphorylated rhodopsin, unactivated cyclic GMP phosphodiesterase, or phosphodiesterase (PDE) activated by GMP PNP-bound G-protein. These data show that although 48-kDa protein has ATPase activity, lack of regulation of this activity by the elements of visual transduction makes it unlikely for this activity to have a role in quenching the light activation of cyclic GMP cascade.

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Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21.

The EMBO Journal ◽

10.1002/j.1460-2075.1984.tb01808.x ◽

1984 ◽

Vol 3 (2) ◽

pp. 339-341 ◽

Cited By ~ 69

Author(s):

R. Leberman ◽

U. Egner

Keyword(s):

Binding Site ◽

Primary Structure ◽

Binding Proteins ◽

Nucleotide Binding ◽

Nucleotide Binding Site ◽

Gtp Binding Proteins ◽

Gtp Binding

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Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(89)91615-x ◽

1989 ◽

Vol 160 (1) ◽

pp. 25-32 ◽

Cited By ~ 36

Author(s):

Paul G. Polakis ◽

Ralph Snyderman ◽

Tony Evans

Keyword(s):

Binding Protein ◽

Nucleotide Binding ◽

Binding Domain ◽

Nucleotide Binding Domain ◽

Gtp Binding Protein ◽

Gtp Binding

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Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

Biochemistry and Cell Biology ◽

10.1139/o86-042 ◽

1986 ◽

Vol 64 (4) ◽

pp. 304-308 ◽

Cited By ~ 3

Author(s):

B. D. Gupta ◽

T. J. Borys ◽

S. Deshpande ◽

R. E. Jones ◽

E. W. Abrahamson

Keyword(s):

Light Scattering ◽

Outer Segment ◽

Plasma Membranes ◽

Binding Protein ◽

Rod Outer Segments ◽

Particle Scattering ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Time Period ◽

Hydrolysis Of

In the presence of exogeneous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition. GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5′-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.

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