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Measurement of GTPγS binding to specific G proteins in membranes using G‐protein antibodies
Author(s) -
Okamoto Takashi,
Ikezu Tsuneya,
Murayama Yoshitake,
Ogata Etsuro,
Nishimoto Ikuo
Publication year - 1992
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(92)80878-k
Subject(s) - gtp' , membrane , antiserum , chemistry , g protein , kinetics , antibody , biochemistry , microbiology and biotechnology , biology , receptor , enzyme , physics , quantum mechanics , immunology
We developed a novel method to quantitatively measure GTPγS binding to specific G proteins in crude membranes using G‐protein antibodies. The basic strategy was that the materials were initially incubated with [ 35 S]GTPγS at 37°C. After 4°C incubation in the wells of an ELISA plate precoated with G‐protein antibodies, the radioactivity of each well was counted. This method, using an anti‐G i antiserum and an anti‐G 3 antiserum, quantitatively and specifically detected the binding of GTPγS to purified G 12 and G 3 . In S49 cell membranes, GTPγS binding to immunoreactive G 3 was observed in a time‐dependent manner that obeyed first order kinetics, and the rate constant was stimulated ∼tworold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTPγS binding to specific G proteins in cell membranes.