Stabilization and solubilization of bovine corpus-luteum adenylate cyclase. The effects of guanosine triphosphate, guanosine 5′-[β,γ-imido]triphosphate, sodium fluoride and Tris/hydrochloric acid concentration on enzyme activity

1. Adenylate cyclase of the washed 600g sediment of bovine corpus-luteum homogenate is stimulated by p[NH]ppG (guanosine 5'-[beta,gamma-imido]triphosphate), the imido analogue of GTP, and to a lesser extent by GTP itself. Activation by p[NH]ppG is not reversed by extensive washing before assay, but can, however, be reversed by NaF. 2. Both p[NH]ppG and NaF stabilize the enzyme during incubation at 37 degrees C. NaF also causes an irreversible activation, but only of part of the potentially NaF-activatable adenylate cyclase; there are possibly two components of the adenylate cyclase system, which can be distinguished by their response to NaF. 3. Solubilization of the adenylate cyclase activity in the 600g sediment, by using the non-ionic detergent Lubrol-PX, gave variable yields. A relationship between the magnitude of NaF stimulation of the 600g-sediment enzyme and the yield of soluble activity derived from the sediment was recognized. The results suggest that the pre-existing state of the enzyme complex in vivo is reflected by the response in vitro to NaF and may determine the success with which activity can be solubilized. 4. The absolute yields of soluble activity could be increased by p[NH]ppG preactivation of the 600g sediment. During the development of the maximally active state by preincubation with p[NH]ppG the enzyme passes through a stage in which Lubrol solubilization is increased, but the maximally active state is itself less amenable to solubilization. p[NH]ppG activation causes the appearance of NaF-inhibited states, which appear to be preferentially solubilized by Lubrol-PX.     

Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976) Circ. Res. 39, 586–595; Engelhard, Plut & Storm (1976) Biochim. Biophys. Acta 451, 48–61]. Further, the results of this study show that cardiac sarcoplasmic-reticulum membranes possess this enzyme.     

Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes.

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45--50-fold increase in cyclic AMP production over 30 min compared with 12--13 fold p[NH[ppG and 2--3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.     

Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors. Evidence for the involvement of a guanine nucleotide regulatory protein.

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.     

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a `burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a `lag' phase with p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.     

Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta.

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

The phorbol ester TPA stimulates the expression of functional beta-adrenoceptors in human T lymphoblasts Molt 3.

In crude membranes from human T lymphoblasts Molt 3 cultured under standard conditions, the adenylate cyclase system was stimulated by GTP, its beta gamma-imido analogue (p[NH]ppG,) NaF and forskolin, but not by isoprenaline, prostaglandin E1 and vasoactive intestinal peptide. TPA (tumour-promoting agent phorbol ester) added at low concentration (3.2 nM) to the culture medium induced a marked increase in functional beta 2-adrenoceptors. Competition curves of [125I]cyanopindolol with the antagonist ICI 118.551 and four beta-adrenergic agonists indicated that the emergence of functional beta 2-adrenoceptors corresponded to one class of binding sites, shifting from a high-affinity state for agonists to a low-affinity state in the presence of p[NH]ppG. This expression of beta 2-adrenoceptors after a 4 h lag period depended on newly formed mRNA and protein synthesis as judged by the inhibitory effects of actinomycin D and cycloheximide. Further effects of TPA included alterations of the stimulatory G-protein Gs and/or the catalytic unit of adenylate cyclase.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Effect of hormone and guanyl nucleotide pretreatment on the activation energy of pancreatic adenylate cyclase.

The activation energy of adenylate cyclase by p[NH]ppG in rat pancreatic plasma membranes was estimated to be 141-189 kj/mol. When a high concentration of secretin or CCK-8 (C-terminal octopeptide of cholecystokinin-pancreozimin) was added to the assay medium, the activation energy was reduced to 73 kj/mol. This hormone effect was exerted on the activation energy of the activation process of adenylate cyclase by p[NH]ppG. Indeed, when plasma membranes were preactivated with p[NH]ppG alone or with p[NH]ppG and CCK-8 and then washed, there resulted a persistent activation with low activation energy (65 and 48 kj/mol, respectively). A similar low activation energy was observed in membranes preincubated with GMP and CCK-8. The latter treatment could not induce persistent activation but facilitated the activation by p[NH]ppG, suggesting that the step of p[NP]ppG activation requiring a high activation energy in the absence of hormone had developed during preincubation with GMP and CCK-8, and had not been reversed by membrane washing. By contrast, EDTA pretreatment did not influence p[NH]ppG activation while provoking a reversible deactivation of persistently activated adenylate cyclase.     

Transient States of Adenylate Cyclase in Brain Membranes

Basal activity of adenylate cyclase from the amygdala of sheep brain and the neostriatum of turkey brain decays in two phases at 37 degrees C. The first phase is rapid (t1/2 = 2.3 +/- 0.3 min) and results in the loss of 60-70% of basal activity. The second phase is slow (t1/2 approximately 100 min) during which time the catalytic units denature irreversibly. The GTP analogue guanosine-5' (beta-gamma imino) triphosphate (p[NH]ppG) prevents the rapid decay by stabilizing the enzyme at its initial level of activity and also reactivates the enzyme to initial levels during or immediately following the early phase, indicating that denaturation of neither the guanylnucleotide units nor the catalytic units causes the rapid decline in basal activity. Activation by p[NH]ppG is rapid at 37 degrees C, but the binding of p[NH]ppG to the guanylnucleotide subunit also occurs at nonactivatory temperatures. This is determined by the protection of catalytic units from thermal or N-ethylmaleimide inactivation after extensive washing. Thus, at 25 degrees C all of the catalytic units can be stabilized by saturating p[NH]ppG concentrations. At 0 degree C, 35% of the catalytic units can be stabilized by saturating p[NH]ppG concentrations within 30 s. The half-saturation constant for the binding of p[NH]ppG at 0 degree C is identical to that derived in an assay at 37 degrees C, or after an incubation of the membranes for 10 min at 45 degrees C, when the process of thermal denaturation is 80% complete (K1/2 approximately 3 +/- 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation and stabilization of the catalytic unit of adenylate cyclase.

The requirements for stability and activity of the catalytic unit (C) of adenylate cyclase were investigated. After solubilization of bovine brain membranes in the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate (Chaps), the catalytic unit was separated from the stimulatory guanine-nucleotide-binding protein (Gs) by gel filtration on Ultrogel AcA-34. The partially purified C unit was rapidly inactivated at 30 degrees C; 0.25 mM-ATP stabilized activity. Although C-unit activity was dependent on Mg2+ or Mn2+, stabilization by ATP did not require bivalent cations. Activity of the Ultrogel-AcA-34-purified C unit was increased by Ca2+ plus calmodulin and by phosphatidylcholine plus lysophosphatidylcholine; activity in the presence of both activators was significantly greater than with each alone. Calmodulin plus Ca2+ and phospholipids also stabilized C unit. The column-purified C unit was activated by forskolin; the effect of forskolin was additive to those of calmodulin plus Ca2+ and phospholipids. p[NH]ppG-stimulated adenylate cyclase activity was reconstituted by mixing samples from the gel-filtration column containing Gs with C unit. Activation by Ca2+ plus calmodulin and Gs plus p[NH]ppG was additive; Ca2+ plus calmodulin did not alter the concentration of p[NH]ppG required for half-maximal activation. Results were similar with forskolin and Gs plus p[NH]ppG; the presence of one activator did not alter the effect of the other. These studies define conditions for separation of C unit and Gs from brain adenylate cyclase and demonstrate that ATP (in the absence of bivalent cations), phospholipids, calmodulin plus Ca2+, and forskolin all interact with C unit in a manner that is independent of functional Gs.     

Properties of adenylate cyclase solubilized from rat adrenal membranes: effects of ACTH and other stimulators on solubilization.

We have solubilized adenylate cyclase in a relatively stable form from rat adrenal membranes. The solubilized enzyme elutes on a column of Sepharose 4BR as a distinct peak with a higher molecular weight than the soluble fractions which bind 125I-ACTH. Both the soluble and membrane bound enzymes are activated by NaF and Gpp(NH)p, and both have similar affinities for MgATP. While the membrane bound enzyme is activated similarly by either Mg2+ or Mn2+, the soluble enzyme is more fully activated by Mn2+. Pretreatment of adrenal membranes with NaF or Gpp(NH)p before the addition of detergent enhances recovery of soluble enzyme activity, while recovery of activity in the unsolubilized membrane pellet is unchanged. In contrast, addition of ACTH prevents solubilization of the enzyme and greatly increases its recovery in the pellet. This observation is consistent with the theory that action of the hormone on a receptor subunit leads to an association between the receptor and a catalytic subunit. Such an association might make it more difficult to remove the enzyme from the surrounding lipid matrix of the membrane.     

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.     

Mechanism of desensitization of adenylate cyclase in lutropin. GTP-dependent uncoupling of the receptor

Lutropin-sensitive adenylate cyclase ((EC 4.6.1.1) ATP pyrophosphate-lyase (cyclizing)) in purified rat ovarian plasma membranes is stimulated by lutropin 2- to 3-fold in the absence, but 15- to 20-fold in the presence of GTP or p(NH)ppG. Following 10 to 15 min of incubation at 30 degrees C in the presence of lutropin, enzyme activity declined (50%) in the presence of GTP but not in the presence of p(NH)ppG. This desensitizing process induced by lutropin and GTP is not seen if NaF is also included in the incubation medium. The desensitized state of the enzyme persists at 4 degrees C in membranes washed free of the incubation medium. In this state the enzyme is characterized by: (i) a reduced response to lutropin even in the presence of p(NH)ppG; (ii) its response to NaF is not different from that of untreated enzyme; (iii) it reconverts to a fully responsive state following incubation (10 min, 30 degrees C) in GTP-free medium, a process accelerated by p(NH)ppG; (iv) the receptor content as well as the stability of the receptor.hormone complex does not differ from that of untreated fully responsive enzyme. It is proposed that desensitization results from a GTP-dependent, hormone-stimulated reaction that leads to impaired coupling of the enzyme system. The desensitized state induced is transient and may revert to a responsive one under specified conditions.     

Association of binding sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Interaction of gastrointestinal hormones

1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.     

Activation of rat liver adenylate cyclase by guanosine 5''-[beta,gamma-imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein.

The effects of guanosine 5'-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi-irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state.     

Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

The kappa-opioid receptor from human placenta: hydrodynamic characteristics and evidence for its association with a G protein

The kappa nature of opioid binding sites in a brush border membrane (BBM) fraction from human placenta has been confirmed: these sites display considerably higher apparent affinity (KI = 1.2 nM) for the kappa selective ligand U-50488 than they do for the mu and delta selective ligands [D-Ala2, MePhe4, Glyol5] enkephalin (KI = 1.5-2 microM) and [D-Thr2, Leu5] enkephalyl-Thr (KI = 10-15 microM), respectively. The BBM fraction from human placenta was incubated either with the agonist 3H-etorphine or with the antagonist 3H-diprenorphine and subsequently solubilized with digitonin. The solubilized macromolecular radioactivity was found to behave as a homogeneous entity both in molecular exclusion chromatography (app. rs = 6.1 nm) and in linear sucrose gradients (app. S20.w = 12 S). Two lines of evidence indicated that the placental kappa opioid receptor is capable of interacting with a guanine nucleotide regulatory (G) protein: (i) equilibrium binding of the agonist 3H-etorphine in the BBM fraction was clearly inhibited by 5'-guanylylimidodiphosphate (Gpp(NH)p), especially in the presence of Na+ ions while binding of the antagonist 3H-diprenorphine was significantly less so and (ii) the sedimentation velocity of the kappa opioid receptor was decreased down to about 10 S when the BBM fraction was prelabeled with radioligand in the presence of Gpp(NH)p prior to its solubilization with digitonin. The G protein that mediates the effect of Gpp(NH)p might be neither Gs nor Gi since no adenylate cyclase activity could be demonstrated in the BBM fraction from human placenta.