Abstract
We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.
Highlights
Such activation occurs via dissociation of the inactive aj3y heterotrimer into the active a and By forms
Chem. 263, 9482-9486) develasby,we have dissociated been and able to establish a quantitativeELISA for to assess the subunit dissociation model opment of antipeptide antibodies to the a. protein of for activation ofG. in native targetcell membranes
Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP andguanosine 6‘-0-(thiotriphosphate) (GTPrS) and the &adrenergic specificity of the antibodies for a,have been previously reported [6] as have methods for purification of antibodies by sequential chromatography of rabbit serum over Sephadex G-25, DEAE-AffiGel blue, and a peptide affinity column
Summary
Such activation occurs via dissociation of the inactive aj3y heterotrimer into the active a and By forms. Were prepared from rabbit liver [8] and used as bdabrisalsenoetcosia.atTciohtinuvsa,itnrede2cic1eap0tt0eormt-hmoaletedcueiaalteceshdodf&iGsas.doircneiannteaiortginviocefmrGee.cmeiiss-ptobcdroeeasEctdLerediIbsScewArdiibtuAhendsdtsheaeeryls-ep“DwReephettaesiirudlesle!tsuaosBnefdrdtiheDafeilssyvca,aunlmstisidgiiacoetrnnioo.,t”niatenorfd the ELISA assay will plates(Falcon) were diluted antibody was likely to be the major site of amplification of signal added to plates in theabsence or presence of purified G., peptide, or transduction by agonists active at hormone receptors sodium cholate (purified as described [9])extracts of cell membranes.
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