Specific binding of leukotriene B4 to guinea pig lung membranes

We have demonstrated binding sites for LTB4 in guinea pig lung membranes. Binding of [3H]-LTB4 was of high affinity (Kd = 0.76 nM), saturable and linear with protein concentration (0.2-1.2 mg/ml). Scatchard and Hill's plot analysis indicated a single class of binding site with a Hill's coefficient of 0.99 +/- 0.08 (n = 4). [3H]-LTB4 was unmetabolized during incubation with membrane preparations, as indicated by high performance liquid chromatography. Divalent cations such as Mg2+ and Ca2+ enhanced binding capacity without changing the Kd. Na+ ions decreased binding in a concentration-dependent manner. Guanine nucleotides, GTP, GTP gamma S and Gpp(NH)p also decreased the number of binding sites. Finally, competition experiments demonstrated the following order of potency for displacement of [3H]-LTB4 from its receptor site: LTB4 greater than 20-OH-LTB4 much greater than 20-COOH-LTB4 = 6-trans-12-epi-LTB4 greater than LTC4 = LTD4 = 5-HETE. These data indicate that a specific LTB4 receptor, in addition to the previously documented LTC4 and LTD4 receptors, exists in guinea pig lung.     

Muscarinic acetylcholine receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes

Receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]), to guanine-nucleotide-binding proteins (G-proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholine (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1-1 microM. In contrast, the GDP analog, guanosine 5'-O-(2-thiodiphosphate), could not replace GDP in promoting carbachol-induced increase in [35S]GTP[S] binding. In addition to GDP, agonist-induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required the presence of Mg2+, being half-maximally and maximally effective at about 30 microM and 300 microM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist-stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5-fold, with half-maximal and maximal increase being observed at 5-10 microM and 100 microM, respectively. The agonist-induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two--three-fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of the membranes with pertussis toxin under conditions leading to 95% ADP-ribosylation of the toxin-sensitive G-protein alpha-subunits markedly reduced agonist-stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist-stimulated binding of [35S]GTP[S] to G-proteins can be a sensitive assay for measuring receptor-regulated G-protein activation in native membranes and, furthermore, suggest that one agonist-activated mACh receptor can activate two or three cardiac G-proteins, being mainly members of the pertussis-toxin-sensitive G-proteins.     

Nucleoside transport in heart: species differences in nitrobenzylthioinosine binding, adenosine accumulation, and drug-induced potentiation of adenosine action

The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep greater than hydroxynitrobenzylthioguanosine greater than dipyridamole greater than hexobendine much greater than lidoflazine much greater than flunitrazepam greater than diazepam greater than lorazepam greater than flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated.(ABSTRACT TRUNCATED AT 250 WORDS)     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.

Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Signal amplification in HL-60 granulocytes. Evidence that the chemotactic peptide receptor catalytically activates guanine-nucleotide-binding regulatory proteins in native plasma membranes

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) [G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal (less than or equal to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (greater than 100-fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive [35S]GTP[S]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

Dissociation of guanosine 5'-[gamma-thio]triphosphate from guanine-nucleotide-binding regulatory proteins in native cardiac membranes. Regulation by nucleotides and muscarinic acetylcholine receptors.

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] greater than GTP greater than or equal to guanosine 5'-[beta,gamma-imino]triphosphate greater than GDP greater than or equal to guanosine 5'-[beta-thio]diphosphate greater than GMP, markedly enhanced the observed dissociation, with 20-30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25 degrees C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetylcholine receptors. Carbachol-stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]-binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTP[S]-bound G proteins, resulting in release of bound guanine nucleoside triphosphate.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins

The V1 vasopressin receptor has been solubilized from rat liver membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate (CHAPS) and reconstituted into phospholipid vesicles. There is essentially complete solubilization of the receptor by 3% CHAPS at a protein concentration of 15 mg/ml. Reconstitution into soybean phospholipid vesicles is readily achieved either by gel filtration chromatography or by membrane dialysis. The binding of [3H]vasopressin to proteoliposomes is specific, saturable, reversible, and magnesium-dependent. In contrast, the detergent-soluble vasopressin receptor does not display specific binding. The apparent affinity of the reconstituted receptor for [3H]vasopressin is approximately 4-fold lower than that of the receptor in native membranes. In addition, the binding of [3H]vasopressin to reconstituted vesicles is not sensitive to 100 microM guanosine 5'-O-thiotriphosphate (GTP gamma S) as it is in native membranes. However, the apparent affinity of the reconstituted receptor for ligand approximates that of native membranes when membranes are prebound with vasopressin prior to solubilization and reconstitution into vesicles. Furthermore, vesicles reconstituted from membranes prebound with vasopressin show GTP gamma S sensitivity of [3H] vasopressin binding. This finding strongly suggests that vasopressin stabilizes a receptor-G-protein complex during solubilization. The rat liver vasopressin receptor is a glycoprotein, as shown by its specific binding to the lectin "wheat germ agglutinin." The vasopressin receptor can be reconstituted from the N-acetylglucosamine-eluted peak of a wheat germ agglutinin-Sepharose column, and [3H] vasopressin binding activity is purified 5-6-fold from membranes by this chromatographic procedure. The functionality of the partially purified receptor is indicated by its ability to bind ligand with high affinity and by its ability to functionally interact with a G-protein when vasopressin is bound prior to solubilization.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

[3H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.