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Reconstitution of cyc − S49 membranes by in vitro translated G sα
Author(s) -
Journot L.,
Bockaert J.,
Audigier Y.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81460-7
Subject(s) - membrane , in vitro , gtp' , cholera toxin , chemistry , adp ribosylation , adenylate kinase , biochemistry , biophysics , microbiology and biotechnology , receptor , biology , enzyme , endocrinology , nad+ kinase
After ADP‐ribosylation by cholera toxin which promotes dissociation of the subunits, the α‐subunit of G s (G sα ) remained strongly associated with plasma membranes of wild‐type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH ≥ 11.5. In vitro translated G sα could interact with plasma membranes from the cyc − mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTPγS did not alter anchorage of G sα . The characteristics of the association of in vitro translated G sα with cyc − membranes after GTPγS treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP‐ribosylated form in wild‐type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc − membranes by in vitro translated α‐subunit of G s .