Kinetic and hydrodynamic properties of transducin: comparison of physical and structural parameters for GTP-binding regulatory proteins.
Transducin is a member of the family of GTP-binding regulatory proteins that interact with cell surface receptors and that include Gs, Gi, and Go. Kinetic and physical properties of purified bovine transducin were characterized by the following results: (1) Initial rate analysis demonstrates a dissociative-type mechanism for the guanine nucleotide exchange process of transducin in the absence of rhodopsin. A second-order rate constant of kf = (1.7-2.7) X 10(-7) M-1 s-1 was determined for this reaction. (2) Equilibrium binding measurements indicated a Kd of 0.05-0.10 microM for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to transducin. (3) Neither the rate nor the extent of GTP gamma S binding was affected in the presence of up to 50 mM Mg2+, as compared to values obtained in the presence of excess ethylenediaminetetraacetic acid. (4) Sucrose density gradient ultracentrifugation gave S20,w values for transducin, its alpha subunit, and its beta gamma subunit complex of 4.23 +/- 0.25, 3.42 +/- 0.37, and 4.04 +/- 0.2, respectively. (5) Incubation of transducin in the presence of up to 20 mM Mg2+ did not alter its sedimentation behavior; however, the presence of guanine nucleotides did produce a shift in transducin's migration in the sucrose gradient. (6) Gel filtration over Sephacryl S-300 indicated that transducin elutes at a Stokes radius of 37.5 A and that transducin's alpha subunit displays a Stokes radius of 24 A. (7) A molecular mass of 68 kDa for transducin is derived from the determined hydrodynamic parameters. These results are compared with properties known for other G proteins, and functional differences between transducin and Gs, Gi, and Go are proposed in relation to the proteins' primary sequences.
D000818:Animals; D002417:Cattle; D003624:Darkness; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism; D015293:Transducin
D006150:Guanine Nucleotides; D008565:Membrane Proteins; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D015293:Transducin