Identification of functionally important cysteines in the alpha-subunit of transducin by chemical cross-linking techniques.
Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, Talpha and Tbetagamma. N,N'-1,2-phenylenedimaleimide (o-PDM) and N,N'-1,4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of Talpha with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and >200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of Tbetagamma with both reagents produced a major species of 46-kDa. The combination of intact Talpha and o-PDM- or p-PDM-treated Tbetagamma reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified Talpha was incubated with intact Tbetagamma, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on Talpha represent functionally important residues of T.
Millán
Enrique J
EJ
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Bubis
José
J
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry; D010655:Phenylenediamines / Q000737:chemistry; D021122:Protein Subunits; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013997:Time Factors; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D010655:Phenylenediamines; D021122:Protein Subunits; D006165:Guanylyl Imidodiphosphate; D015293:Transducin; D003545:Cysteine