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A high affinity GTP binding site in rat brain
European Journal of Pharmacology, 64 (1980) 365--366
365
© Elsevier/North-Holland Biomedical Press
Rapid communication A HIGH AFFINITY GTP BINDING SITE IN RAT BRAIN JACK E. ROSENBLATT *, REBECCA DEL CARMEN and RICHARD JED WYATT
Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, National Institute o f Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A. Received 12 M a y 1980, accepted 13 M a y 1980
Increasing evidence suggests the involvement of GTP in the activation of adenylate cyclase (Levitzki, 1976). Although the mechanism by which GTP mediates agonist-induced adenylate cyclase activation is unknown, it is thought to involve the binding of GTP to a specific guanine nucleotide-binding protein associated with the cell membrane hormone receptor (Rodbell, 1980). In the present study we demonstrate the presence of specific high affinity binding sites for 3H-GTP in rat caudate. Caudates (35 mg each) from male SpragueDawley rats (175--200 g) were homogenized (Brinkman Polytron, Model PT10, setting 7 for 30 sec) in 100 volumes (w/v) of ice-cold 0.05 M Tris 7.7, and centrifuged at 30,000 × g for 10 min; this washing procedure was repeated once, and the final tissue pellet suspended in 115 volumes of 0.05 M Tris buffer containing 120 mM NaC1, 5 mM KC1, 1 mM MgC12, pH 7.2 at 37°C. 3H-GTP binding was determined by incubating aliquots of the membrane suspension (final concentration 7 mg original wet weight/ml) with 3H-GTP (42 Ci/mmole, New England Nuclear) and, where appropriate, other drugs or nucleotides in a final volume of 1 ml for 10 min at 0°C. Following incubation, the samples were rapidly filtered under vacuum through Whatman GF/B glass fiber filters with three 5-ml washes of cold 0.05 M Tris 7.7 buffer. Radioactivity trapped on the filters was counted after stand* To w h o m correspondence should be addressed.
ing overnight in 10 ml Aquasol (New England Nuclear) at an efficiency of 45%. Since maximal inhibition of 3H-GTP binding first occurred in the presence of 10-4M unlabelled GTP, specific binding was defined as that displaced by 10-4M GTP and represented 90--98% of the total binding at 0.25-25.0 nM 3H-GTP. Scatchard analysis of GTP-displaceable 3HGTP binding indicated two populations of binding sites (fig. 1): a higher affinity lower capacity site (Kd = 2.0 nM, Bmax = 164 pmol/ g caudate), and a lower affinity higher capacity binding site (Kd = 13 nM, Bmax = 690 pmol/g caudate). Association of 3H-GTP was rapid with maximum binding occurring by 5 min; following addition of 10 -4 M unlabelled GTP at 10 min of incubation, dissociation was biphasic; dissociation of 77% of bound 3H-GTP occurred by 15 min, followed by a slower dissociation of an additional 10% over the ensuing 45 min. The amount of 3H-GTP specifically bound was the same in the absence of ions as in the presence of monovalent and divalent cations and chloride ion. Exposure of caudate membranes to 53°C for 15 min caused loss of over 90% of 3H-GTP binding. The specific binding of 3H-GTP was not displaced by ATP in concentrations as high as 1 mM. Cytosine triphosphate (CTP) displaced 3H-GTP (ICs0 = 13 nM), but was more than 100-fold less potent in displacing 3H-GTP than GTP itself. Guanosine diphosphate (GDP) was equipotent with GTP in displacing
366
50C 40C
,..~'.300 ~
•
o 200 I0[
,oo 200 600 1000
2000 3000 Bound(fmoles/ml)
4000
5000
Fig. 1. 0.25--25.0 nM 3H-GTP was incubated with rat caudate membranes f()r 10 rain at 0°C. Free and membrane-bound 3H-GTP were separated by filtration following incubation. Each point representsthe mean of triplicatedeterminations in a singleexperiment. The apparent dissociation constant Kd and binding site density, Bmax, were determined graphically by Scatchard analysis. Similar results were obtained in three separate experiments.
3H-GTP specific binding. Neither guanine nor guanosine displaced 3H-GTP binding. Also dopamine and haloperidol in concentrations
up to I00 p M failed to displace 3H-GTP specific binding. Following incubation with 1.3 n M 3H-GTP, similar amounts of specific binding (50--60 pmol/g original weight) were observed in cerebral cortex, hippocampus, caudate, cerebellum, hypothalamus and medulla-pons. Although the biological relevance of G T P binding is not known, it is possible that a proportion of high affinity caudate binding of 3H-GTP is occurring to the nucleotide binding protein associated with cell m e m b r a n e neurotransmitter receptors. Thus, the study of high affinity SH-GTP binding m a y offer a n e w approach to the study of interactions among neurotransmitters, cell m e m b r a n e receptors and adenylate cyclase activation in brain.
References Levitzki, A., 1976, The mode of coupling of adenylate cyclase to hormone receptors and its modulation by GTP, Biochem. Pharmacol. 27, 2083. Rodbell, M., 1980, The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature 284, 17.
365
© Elsevier/North-Holland Biomedical Press
Rapid communication A HIGH AFFINITY GTP BINDING SITE IN RAT BRAIN JACK E. ROSENBLATT *, REBECCA DEL CARMEN and RICHARD JED WYATT
Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, National Institute o f Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A. Received 12 M a y 1980, accepted 13 M a y 1980
Increasing evidence suggests the involvement of GTP in the activation of adenylate cyclase (Levitzki, 1976). Although the mechanism by which GTP mediates agonist-induced adenylate cyclase activation is unknown, it is thought to involve the binding of GTP to a specific guanine nucleotide-binding protein associated with the cell membrane hormone receptor (Rodbell, 1980). In the present study we demonstrate the presence of specific high affinity binding sites for 3H-GTP in rat caudate. Caudates (35 mg each) from male SpragueDawley rats (175--200 g) were homogenized (Brinkman Polytron, Model PT10, setting 7 for 30 sec) in 100 volumes (w/v) of ice-cold 0.05 M Tris 7.7, and centrifuged at 30,000 × g for 10 min; this washing procedure was repeated once, and the final tissue pellet suspended in 115 volumes of 0.05 M Tris buffer containing 120 mM NaC1, 5 mM KC1, 1 mM MgC12, pH 7.2 at 37°C. 3H-GTP binding was determined by incubating aliquots of the membrane suspension (final concentration 7 mg original wet weight/ml) with 3H-GTP (42 Ci/mmole, New England Nuclear) and, where appropriate, other drugs or nucleotides in a final volume of 1 ml for 10 min at 0°C. Following incubation, the samples were rapidly filtered under vacuum through Whatman GF/B glass fiber filters with three 5-ml washes of cold 0.05 M Tris 7.7 buffer. Radioactivity trapped on the filters was counted after stand* To w h o m correspondence should be addressed.
ing overnight in 10 ml Aquasol (New England Nuclear) at an efficiency of 45%. Since maximal inhibition of 3H-GTP binding first occurred in the presence of 10-4M unlabelled GTP, specific binding was defined as that displaced by 10-4M GTP and represented 90--98% of the total binding at 0.25-25.0 nM 3H-GTP. Scatchard analysis of GTP-displaceable 3HGTP binding indicated two populations of binding sites (fig. 1): a higher affinity lower capacity site (Kd = 2.0 nM, Bmax = 164 pmol/ g caudate), and a lower affinity higher capacity binding site (Kd = 13 nM, Bmax = 690 pmol/g caudate). Association of 3H-GTP was rapid with maximum binding occurring by 5 min; following addition of 10 -4 M unlabelled GTP at 10 min of incubation, dissociation was biphasic; dissociation of 77% of bound 3H-GTP occurred by 15 min, followed by a slower dissociation of an additional 10% over the ensuing 45 min. The amount of 3H-GTP specifically bound was the same in the absence of ions as in the presence of monovalent and divalent cations and chloride ion. Exposure of caudate membranes to 53°C for 15 min caused loss of over 90% of 3H-GTP binding. The specific binding of 3H-GTP was not displaced by ATP in concentrations as high as 1 mM. Cytosine triphosphate (CTP) displaced 3H-GTP (ICs0 = 13 nM), but was more than 100-fold less potent in displacing 3H-GTP than GTP itself. Guanosine diphosphate (GDP) was equipotent with GTP in displacing
366
50C 40C
,..~'.300 ~
•
o 200 I0[
,oo 200 600 1000
2000 3000 Bound(fmoles/ml)
4000
5000
Fig. 1. 0.25--25.0 nM 3H-GTP was incubated with rat caudate membranes f()r 10 rain at 0°C. Free and membrane-bound 3H-GTP were separated by filtration following incubation. Each point representsthe mean of triplicatedeterminations in a singleexperiment. The apparent dissociation constant Kd and binding site density, Bmax, were determined graphically by Scatchard analysis. Similar results were obtained in three separate experiments.
3H-GTP specific binding. Neither guanine nor guanosine displaced 3H-GTP binding. Also dopamine and haloperidol in concentrations
up to I00 p M failed to displace 3H-GTP specific binding. Following incubation with 1.3 n M 3H-GTP, similar amounts of specific binding (50--60 pmol/g original weight) were observed in cerebral cortex, hippocampus, caudate, cerebellum, hypothalamus and medulla-pons. Although the biological relevance of G T P binding is not known, it is possible that a proportion of high affinity caudate binding of 3H-GTP is occurring to the nucleotide binding protein associated with cell m e m b r a n e neurotransmitter receptors. Thus, the study of high affinity SH-GTP binding m a y offer a n e w approach to the study of interactions among neurotransmitters, cell m e m b r a n e receptors and adenylate cyclase activation in brain.
References Levitzki, A., 1976, The mode of coupling of adenylate cyclase to hormone receptors and its modulation by GTP, Biochem. Pharmacol. 27, 2083. Rodbell, M., 1980, The role of hormone receptors and GTP-regulatory proteins in membrane transduction, Nature 284, 17.