@article{3600129048f54164930dd75a978b19ad,
title = "Measurement of GTPγS binding to specific G proteins in membranes using G-protein antibodies",
abstract = "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 [35S]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-Gi antiserum and an anti-G3 antiserum, quantitatively and specifically detected the binding of GTPγS to purified G12 and G3. In S49 cell membranes, GTPγS binding to immunoreactive G3 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.",
keywords = "Antibody, Crude membrane, ELISA plate, G protein, G protein activity, GTPγS binding",
author = "Takashi Okamoto and Tsuneya Ikezu and Yoshitake Murayama and Etsuro Ogata and Ikuo Nishimoto",
note = "Funding Information: Acknowledgements: We thank Drs. Toshiaki Katada (Tokyo Institute of Tcehnolo~gy)a nd Toshlhlde Nukada (University of Tokyo) for their Sifts of (3 proteins and O,a eDNA; and Drs. Koji lshii, Atsushi Imaizumi, and Yoshiharu Matsuura for the baeulovirus encoding G,~ eDNA. We are also indebted to Dr. Tatsuya Haga for his gift of $49 cells; Dr. Kazuyoshi Yonezawa (Kobe University) for his indisl~nsa-ble advice; Miss Yasuko Homma and Mrs. Misaki Nagashlma (University of Tokyo) for their technical assistance; Ms. Patricia ll¢¢kman for English correction; and Atomic Research (jeneral Center (Tokyo University) for the use of their facilities. This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, Toyo Jozo Co., Teijin Institute for Biomedical Research, Life Insurance Association of Japan, Napolex Corporation, Japan Research Foundation for Clinical Pharmacology, the Sagavca Foundation for Promotion of Cancer Research, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Arima Memorial Foundation for Medical Research, Japan Heart Foundation Research Grant for 1991, and Kudo Science Foundation.",
year = "1992",
month = jun,
day = "29",
doi = "10.1016/0014-5793(92)80878-K",
language = "English (US)",
volume = "305",
pages = "125--128",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "2",
}