Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.
Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.
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Unité Associée N. 520 du Centre National de la Recherche Scientifique, Centre d'Etudes Nucléaires de Grenoble, France.
Catty
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Bennett
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000378:metabolism; D013388:Succinimides; D015293:Transducin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D013388:Succinimides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); C035760:bis(sulfosuccinimidyl)suberate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins; D015293:Transducin