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1.
Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [alpha(32)P]-GTP. Prior to this study the number of GTP-binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP-binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [alpha(32)P]-GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [alpha(32)P]-GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP-binding proteins. The GTP-binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP-binding proteins in the development and physiology of the placenta.  相似文献   

2.
《FEBS letters》1986,200(1):156-160
Major cytoplasmic GTP-binding proteins in Dictyostelium discoideum were identified by direct photoaffinity labeling with [α-32P]GTP. Three actin-binding proteins and a protein with an apparent molecular mass of 24 kDa (p24) could be labeled with [α-32P]GTP. p24 binds to DEAE-cellulose, behaves like a monomer during gel filtration and was purified to homogeneity by GTP-affinity chromatography. In comparison to other nucleotide triphosphates the binding of GTP to p24 is highly specific.  相似文献   

3.
Abstract

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [125I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [35S]GTPγS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [α-32P]-azidoanilido GTP ([α-32P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Gα-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Gαo, Gαq/11, and Gα. While hSPR's ability to associate with Gq/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Gαo and Gαs.  相似文献   

4.
ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.  相似文献   

5.
As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.  相似文献   

6.
When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.  相似文献   

7.
Exocytosis of the sperm acrosome is an obligate precursor to successful egg penetration and subsequent fertilization. In most mammals, acrosomal exocytosis occurs at a precise time, after sperm binding to the zona pellucida of the egg, and is induced by a specific component of the zona pellucida. It may be considered an example of regulated secretion with the acrosome of the sperm analogous to a single secretory vesicle. Monomeric G proteins of the rab3 subfamily, specifically rab3a, have been shown to be important regulators of exocytosis in secretory cells, and we hypothesized that these proteins may regulate acrosomal exocytosis. Using α[32P] GTP binding to Immobilon blotted mouse sperm proteins, the presence of three or more monomeric GTP binding proteins was identified with Mr = 22, 24, and 26 × 103. Alpha[32P] GTP binding could be competed by GTP and GDP, but not GMP, ATP, or ADP. Anti‐peptide antibodies specific for rab3a were used to identify the 24 kDa G protein as rab3a. Using immunocytochemistry, rab3a was localized to the head of acrosome‐intact sperm and was lost during acrosomal exocytosis. It was identified in membrane and cytosolic fractions of sperm with the predominant form being membrane‐bound, and its membrane association did not change upon capacitation. Immunogold labeling and electron microscopy demonstrated a subcellular localization in clusters to the periacrosomal membranes and cytoplasm. These data identify the presence of rab3a in acrosomal membranes of mouse sperm and suggest that rab3a plays a role in the regulation of zona pellucida ‐induced acrosomal exocytosis. Mol. Reprod. Dev. 53:413–421, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

8.
Abstract: With [3H]guanosine triphosphate ([3H]GTP) and [3H]β, γ -imidoguanosine 5′-triphosphate ([3H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (KD= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [3H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [3H]GTP and [3H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [3H]GTP (into [3H]guanosine) and the likely formation of Ca-guanine nucleotide2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [3H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [3H]GTP and [3H]GppNHp catabolism (into [3H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [3H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [3H]5-hydroxytryptamine ([3H]5-HT) in the rat brain.  相似文献   

9.
Interaction of small G proteins with photoexcited rhodopsin   总被引:1,自引:0,他引:1  
Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the alpha-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.  相似文献   

10.
The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.  相似文献   

11.
Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).  相似文献   

12.
A major 27 kDa particulate and a minor 24 kDa cytosolic GTP-binding protein was detected in HEL cells upon incubation with [-32P]GTP of nitrocellulose blots containing polypeptides separated using SDS-PAGE. Addition of lovastatin (30 M) to HEL cells in culture inhibited protein synthesis by 35%. However, this treatment resulted in a 5-fold increase, as quantitated by [-32P]GTP binding, in the amount of cytosolic 24 kDa GTP-binding protein. Addition of cycloheximide plus lovastatin to cells in culture abolished the observed increase in 24 kDa GTP-binding protein. Incubation of cells with lovastatin plus [R,S]-[5-3H]mevalonolactone resulted in the incorporation of radioactivity into several polypeptides in both the cytosolic and particulate fractions including a polypeptide of molecular mass of 24 kDa in the cytosol. The mobility of this 24 kDa isoprenylated protein on SDS-PAGE was identical to that of the GTP-binding protein increased in response to lovastatin. However, the 24 kDa protein remained in the cytosol after undergoing isoprenylation. The 24 kDa protein was distinct from the HEL cell, G25K/CDC42Hs GTP-binding protein and the GTP-binding protein that was a substrate for botulinum toxin C3 catalyzed ADP-ribosylation. Results demonstrate that lovastatin specifically increases the expression of a 24 kDa GTP-binding protein in HEL cells and that, isoprenylation of low molecular mass GTP-binding protein(s) may have function(s) in addition to its role in the targetting of these proteins to cell membrane.  相似文献   

13.
Detection of G Proteins in Purified Bovine Brain Myelin   总被引:5,自引:5,他引:0  
Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
GTP-binding proteins (GTPases) have been detected in the mitochondria of human placenta. It has been proposed that porin interacts with GTPases in the mitochondrion to modulate contact site function, however, their identity and location is not known. In this study, we investigated the location of GTPases in mitochondria from term placentae as well as the expression of mitochondrial GTPases in mid-term placentae. Mitochondria obtained from human term and mid-term placentae were purified by sedimentation. Sub-mitochondrial vesicles prepared from ruptured and sonicated mitochondria were separated by ultracentrifugation in sucrose density gradients. The location of membrane vesicles was determined using marker enzymes. Mitochondrial proteins were separated by SDS-PAGE. Western blots were incubated in [alpha-(32)P]-GTP and detected using autoradiography or antibodies against known GTPases and porin followed by enhanced chemiluminescence. [alpha-(32)P]-GTP bound 24 and 28 kDa proteins located in the outer membrane. The G(salpha)antibody detected 42.5, 53 and 67 kDa proteins. The G(ialpha)antibody identified a 40.5 kDa band in contact sites and the outer membrane, as well as 55 and 105 kDa proteins in contact site vesicles. The Ran antibody detected a 28 kDa protein, mainly in the outer membrane. Porin migrated at 30 kDa. G(ialpha)and Ran were detected in mitochondria from both term and mid-term placentae. The location of porin and GTPases leave open the possibility that these proteins interact in contact sites and may also be responding to extra-mitochondrial signals. Ran and G(ialpha)are expressed by mid-term in human placentae and may be necessary for placental functions at this stage of development. It will be important in future experiments to characterise the physiological functions of these GTP-binding proteins in the mitochondria of human placenta.  相似文献   

15.
The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine-rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive-like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha-32P]GTP. The reaction was blocked by KA, glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a peptide containing the glycine-rich motif. Site-directed mutagenesis of residues K72 and Y73 within the glycine-rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine-rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine-rich motif is the binding site at which guanine nucleotides inhibit the glutamate-mediated responses of various members of the subfamily of glutamate ionotropic receptors.  相似文献   

16.
G Swarup  D L Garbers 《Biochemistry》1983,22(5):1102-1106
Porcine rod outer segment (ROS) proteins were phosphorylated in the presence of [gamma-32P]ATP and Mg2+, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. The phosphorylation of rhodopsin, the major protein-staining band (Mr approximately 34 000-38 000), was markedly and specifically increased by exposure of rod outer segments to light; various guanine nucleotides (10 microM) including GMP, GDP, and GTP also specifically increased rhodopsin phosphorylation (up to 5-fold). Adenine nucleotides (cyclic AMP, AMP, and ADP at 10 microM) and 8-bromo-GMP (10 microM) or cyclic 8-bromo-GMP (10 microM) had no detectable stimulatory effect on rhodopsin phosphorylation. GTP increased the phosphorylation of rhodopsin at concentrations as low as 100 nM, and guanosine 5'-(beta, gamma-imidotriphosphate), a relatively stable analogue of GTP, was nearly as effective as GTP. Maximal stimulation of rhodopsin phosphorylation by GTP was observed at 2 microM. GMP and GDP were less potent than GTP. Both cyclic GMP and GMP were converted to GTP during the time period of the protein phosphorylation reaction, suggestive of a GTP-specific effect. Transphosphorylation of guanine nucleotides by [32P]ATP and subsequent utilization of [32P]GTP as a more effective substrate were ruled out as an explanation for the guanine nucleotide stimulation. With increasing concentrations of ROS proteins, the phosphorylation of rhodopsin was nonlinear, whereas in the presence of GTP (2 microM) linear increases in rhodopsin phosphorylation as a function of added ROS protein were observed. These results suggest that GTP stimulates the phosphorylation of rhodopsin by ATP and that a GTP-sensitive inhibitor (or regulator) of rhodopsin phosphorylation may be present in ROS.  相似文献   

17.
Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.  相似文献   

18.
Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.  相似文献   

19.
R P Bhullar  R J Haslam 《FEBS letters》1988,237(1-2):168-172
The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.  相似文献   

20.
The effects of guanine nucleotides on binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX), a highly selective A1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [3H]DPCPX in a concentration-dependent manner. The rank order of potency for different guanine nucleotides for increasing [3H]DPCPX binding was the same as for guanine nucleotide-induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., Gi, in the regulation of antagonist binding is suggested. This was confirmed by inactivation of Gi by N-ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [3H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP- or NEM-induced increase in antagonist binding was not caused by an affinity change of A1 receptors for [3H]DPCPX but by an increased Bmax value. Guanine nucleotides had similar effects on membrane-bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high-affinity state for agonists, only a few receptors are labeled by [3H]DPCPX. It is suggested that [3H]DPCPX binding is inhibited when receptors are coupled to Gi. Therefore, uncoupling of A1 receptors from Gi by guanine nucleotides or by inactivation of Gi with NEM results in an increased antagonist binding.  相似文献   

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