Reconstitution of the vertebrate visual cascade using recombinant heterotrimeric transducin purified from Sf9 cells.
For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (T alpha beta gamma) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant T alpha with GTP gamma S. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTP gamma S-loaded recombinant transducin (T(*)) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T(*) was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T(*) suggested that only the complex of PDE with two T(*) molecules, PDE. T(2)(*), was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T(*) in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin.
Min
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KC
Laboratory of Molecular Biology and Biochemistry, Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, New York 10021, USA.
Gravina
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SA
Sakmar
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TP
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012996:Solutions; D018411:Spodoptera; D015293:Transducin / Q000235:genetics / Q000378:metabolism*; D014785:Vision, Ocular / Q000502:physiology*
D011994:Recombinant Proteins; D012996:Solutions; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin