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.     

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.     

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 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.     

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)     

Desensitization of tumour Leydig cells by lutropin: evidence for uncoupling of the lutropin receptor from the guanine nucleotide-binding protein

Purified rat Leydig tumour cells were pretreated with lutropin and the effect on the subsequent response to lutropin was determined. Maximal cyclic AMP production was achieved with the same concentration of lutropin in control and lutropin-pretreated cells; however, the maximum stimulated level in pretreated cells was only 30% of controls. The sensitivity to lutropin was decreased in lutropin-pretreated cells [ED(50) (dose that produces a response that is 50% of the maximum response) 60+/-5.7ng/ml and 8+/-1.8ng/ml (mean+/-s.d., n=3) for controls], as was the rate of maximal cyclic AMP production (0.58, compared with 1.89pmol/10(6) cells per min for controls). However, cholera-toxin-stimulated cyclic AMP production was not decreased by lutropin pretreatment, and a potentiation was seen at all time points studied (up to 6h). Pre-incubation with lutropin caused a decrease in specific (125)I-labelled human choriogonadotropin binding; however, this decrease was abolished if the cells were washed under acidic conditions (pH3.0 for 2min at 4 degrees C), indicating that occupation but not loss of the lutropin receptors had taken place. The effect of pretreating the cells with lutropin on adenylate cyclase activity in purified plasma membranes was also investigated. In plasma membranes from control cells both guanosine 5'-[beta,gamma-imido]triphosphate [p(NH)ppG] plus lutropin and NaF plus lutropin caused a 50-60-fold linear increase in cyclic AMP production over 40min compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone. In plasma membranes isolated from lutropin-treated cells the NaF-plus-lutropin- 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. In contrast the plasma membranes from dibutyryl cyclic AMP-treated cells had similar cyclic AMP production rates to control cells with all stimulants studied. The present evidence obtained from studies both with intact cells and with isolated plasma membranes indicates that the initial lutropin-induced desensitization of the rat Leydig tumour cell is due to a lesion in the hormone-receptor coupling to the guanine nucleotide regulatory protein. This process is apparently not mediated by cyclic AMP.     

Modulation of guanine nucleotide affinity does not affect the first order rate constant of activation of adenylate cyclase in native membranes

A new method was developed to follow the rate of activation of adenylate cyclase in rat brain membranes by rapid freezing and N-ethylmaleimide treatment at 0 degrees C. This method was used to investigate the relationship between the rate of activation of adenylate cyclase by p(NH)ppG and GTP gamma S and their apparent affinities. These studies established the following. 1) The kinetics of activation by p(NH)ppG and GTP gamma S were indistinguishable although the apparent affinity of p(NH)ppG was 20-fold lower than the affinity of GTP gamma S. Activation was first order, kobs varying approximately 1.5-fold (average t 1/2 = 3.5 min, 30 degrees C) between 20-90% occupancy by either guanine nucleotide. 2) Final levels of activity were strictly dependent on the concentration of the nucleotides in a saturable manner. 3) Mg2+ increased the apparent affinity of either guanine nucleotide by 10-20-fold between 0.1 microM and 3 mM free Mg2+ in the presence of 2 mM EDTA but did not enhance the rate or maximal extent of activation. 4) The effects of Mg2+ were expressed through two independent classes of sites with affinities in the nanomolar and micromolar range. 5) A Mg2+ X guanine nucleotide complex was not the substrate for activation. The affinity of Mg2+ for nucleotides was determined as 6.25 mM GTP gamma S, 0.930 mM GTP, 0.156 mM p(NH)ppG. 6) Full activation by p(NH)ppG was completely reversible but activation by GTP gamma S was only partially reversible. These results suggest that: activation of adenylate cyclase in native membranes does not require Mg2+ or irreversible binding of the guanine nucleotide and there are two independent pathways for formation of active adenylate cyclase. A minimal mechanism for activation is discussed in light of current models.     

Insulin affects the ability of Gi to be ADP-ribosylated but does not elicit its phosphorylation in intact hepatocytes

Insulin inhibited the ability of activated pertussis toxin to catalyse the ADP-ribosylation of alpha-Gi in isolated plasma membranes in either the absence of added guanine nucleotides or in the presence of GTP. In contrast, when the non-hydrolysable GTP analogue guanylyl-5'-imido-diphosphate (p[NH]ppG) was added to ribosylation mixtures, to inhibit the action of pertussis toxin in catalysing the ADP-ribosylation of alpha-Gi, then the addition of insulin attenuated the action of p[NH]ppG causing an increase in alpha-Gi ribosylation. Pre treatment of intact hepatocytes with insulin had no effect on the subsequent ability of thiol-preactivated pertussis toxin to cause the ADP-ribosylation of alpha Gi using isolated membranes from such cells. The ability of p[NH]ppG to inhibit forskolin-stimulated adenylate cyclase activity was attenuated in the presence of insulin. Insulin did not cause the phosphorylation of alpha-Gi in either intact hepatocytes or in isolated membranes.     

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.     

Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.     

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.     

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.     

Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, ¹²⁵I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and ¹²⁵I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.     

Solubilization of bovine corpus-luteum adenylate cyclase in lubrol-PX, triton X-100 or digitonin and the stabilizing effect of sodium fluoride present in the solubilization medium.

1. Adenylate cyclase activity of the washed 600g sediment of bovine corpus-luteum homogenate was solubilized by Lubrol-PX, Triton X-100 and digitonin. Digitonin was the least destructive of NaF-stimulated activity. 2. NaF, present in the solubilization medium together with MgSO4, increased the percentage yields of soluble activity from untreated 600g sediment and 600g sediment which had been preincubated with p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate). The stabilizing influence of NaF was most marked with digitonin. However, the highest specific activities of soluble enzyme were obtained with Lubrol-PX as solubilizing agent, since digitonin solubilized more membrane protein than does Lubrol-PX, and less of the activity of the digitonin-dispersed 600g sediment was recovered in the 105000g supernatant. 3. p[NH]ppG also has a stabilizing effect when present during the solubilization, but less so than NaF. 4. Both NaF and MgSO4 alone have a stabilizing effect during solubilization. The greatest amounts of soluble activity were obtained with both agents present in the solubilization medium, there being a synergistic effect.     

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.     

On the mechanism of activation of fat cell adenylate cyclase by guanine nucleotides. An explanation for the biphasic inhibitory and stimulatory effects of the nucleotides and the role of hormones.

Adenylate cyclase activity in purified plasma membranes from rat fat cells displays transient kinetic characteristics in the absence and presence of guanyl=5'=yl imidodiphosphate (Gpp(NH)p). Gpp(NH)p causes immediate inhibition of enzyme activity; the inhibitory phase is followed by a slow increase in activity which, depending on incubation temperature, exceeds activity stimulated in the presence of hormones (glucagon, secretin, epinephrine, or adrenocorticotropin). Basal activity displays an initial high rate of activity which decays to a low state of activity within 2 min of incubation. Hormones do not alter the initial rate but prevent the decay in enzyme activity. The inhibitory phase of Gpp(NH)p action and the previously reported (Harwood, J.P., Low, H., and Rodbell, M. (1973) J. Biol. Chem. 248, 6239-6245) inhibitory effects of GTP are abolished by increasing (Mg2+) and pH to 50 mM and 8.5, respectively. Under these conditions, Gpp(NH)p and GTP cause marked stimulation of activity, the stimulatory effect of Gpp(NH)p being greater than that of GTP both in the absence and presence of hormones...     

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.     

Reexamination of the role of nonhydrolyzable guanosine 5'-triphosphate analogues in tubulin polymerization: reaction conditions are a critical factor for effective interactions at the exchangeable nucleotide site

Recently it was proposed [O'Brien, E. T., & Erickson, H. P. (1989) Biochemistry 28, 1413-1422] that tubulin polymerization supported by guanosine 5'-(beta,gamma-imidotriphosphate) [p(NH)ppG], guanosine 5'-(beta,gamma-methylenetriphosphate) [p(CH2)ppG], and ATP might be due to residual GTP in reaction mixtures and that these nucleotides would probably support only one cycle of assembly. Since we had observed polymerization with these three compounds, we decided to study these reactions in greater detail in two systems. The first contained purified tubulin and a high concentration of glycerol, the second tubulin and microtubule-associated proteins (MAPs). In both systems, reactions supported by nucleotides other than GTP were most vigorous at lower pH values. In the glycerol system, repeated cycles of polymerization were observed with ATP and p(CH2)ppG, but not with p(NH)ppG. With p(NH)ppG, a single cycle of polymerization was observed, and this was caused by contaminating GTP. In the MAPs system, repeated cycles of polymerization were observed with both nonhydrolyzable GTP analogues, even without contaminating GTP, but ATP was not active at all in this system. Binding to tubulin of p(NH)ppG, p(CH2)ppG, and, to a lesser extent, ATP was demonstrated indirectly, since high concentrations of the three nucleotides displaced radiolabeled GDP originally bound in the exchangeable site, with p(NH)ppG the most active of the three compounds in this displacement assay. The failure of GTP-free p(NH)ppG to support tubulin polymerization in our glycerol system even though it displaced GDP from the exchangeable site was further investigated by examining the effects of p(NH)ppG on polymerization and polymer-bound nucleotide with low concentrations of GTP. The two nucleotides appeared to act synergistically in supporting polymerization, so that a reaction occurred with a subthreshold GTP concentration if p(NH)ppG was also in the reaction mixture. Analysis of radiolabeled exchangeable-site nucleotide in polymers formed in reaction mixtures containing both GTP and p(NH)ppG demonstrated that p(NH)ppG which entered polymer did so primarily at the expense of GDP originally bound in the exchangeable site rather than at the expense of GTP. It appears that in the glycerol reaction condition, tubulin-p(NH)ppG cannot initiate tubulin polymerization but that it can participate in polymer elongation. ATP and p(CH2)ppG also entered the exchangeable site during polymerization without GTP in glycerol, as demonstrated by displacement of radiolabeled GDP from polymer when these alternate nucleotides were used.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of fluoride on the state of aggregation of adenylate cyclase in rat liver plasma membranes.

Irradiation inactivation was used to monitor changes in the state of adenylate cyclase in rat liver plasma membranes in the presence of F-.F- caused a decrease in the target size from 328000 to 237000 at 0 degrees C and from 329000 to 219000 at 30 degrees C. Adenylate cyclase was activated by F- at both 0 degrees C and 30 degrees C. The effect of F- was biphasic, activating up to a concentration of 10mM and inhibiting at higher concentrations. If adenylate cyclase weas maximally activated with glucagon and p[NH]ppG ([beta gamma-imido]GTP) all concentrations of F- were inhibitory. The implications of the results with respect to the mechanism of activation of adenylate cyclase are discussed.     

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.