Activation of adenylate cyclase in human platelet membranes by guanosine 5''-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

Incubation of platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate causes a slow increase in GS (stimulatory GTP-binding protein) activation of adenylate cyclase. Mg2+ is necessary for this slow activation. This process is inhibited in the presence of ATP, and inhibition is greater if cyclic AMP is also included in the incubation. Adenosine 5'-[beta gamma-imido]triphosphate instead of ATP in the incubation facilitates the slow activation in the presence of cyclic AMP, and incubation of membranes with cyclic-AMP-dependent protein kinase inhibitor decreased inhibition of the slow activation of adenylate cyclase by ATP and cyclic AMP. A protein of 45 kDa in platelet membranes is phosphorylated in a cyclic-AMP-dependent manner. The transition from a reversibly activated form of GS to an irreversibly activated form is substantially slower in the presence of ATP and cyclic AMP. We propose that guanosine 5'-[beta gamma-imido]triphosphate-activated GS may exist in phosphorylated or non-phosphorylated forms, and that the non-phosphorylated form is the more active of the two species. The non-phosphorylated form of GS may correspond to the irreversibly activated state.     

Similarities and differences in phorbol ester- and luteinizing-hormone-induced desensitization of rat tumour Leydig-cell adenylate cyclase.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was shown to mimic luteinizing hormone (LH; lutropin) in causing desensitization of LH-mediated cyclic AMP production in tumour Leydig cells. However, there were differences between LH- and TPA-induced desensitization: (1) TPA induced a more rapid effect than LH; (2) adenosine did not inhibit TPA-induced desensitization, whereas it completely inhibited the LH-induced desensitization; (3) adenylate cyclase activity in plasma membranes from TPA-desensitized cells was not decreased, whereas similar preparations from LH-desensitized cells lost their response to LH and to LH plus guanosine 5'-[beta gamma-imido]triphosphate; TPA-, but not LH-, treated cells had a decreased capacity to respond to cholera toxin and forskolin. These results indicate that LH and phorbol esters induce desensitization of adenylate cyclase in rat tumour Leydig cells by different mechanisms.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.     

Stimulation of Ca2+ efflux from sarcoplasmic reticulum by preincubation with ATP and inorganic phosphate.

Preincubation of sarcoplasmic reticulum with 1 mM-ATP completely inhibits Ca2+ accumulation and stimulates ATPase activity by over 2-fold. This effect of ATP is obtained only when the preincubation is carried out in the presence of Pi, but not with arsenate, chloride or sulphate. The inhibition by ATP of Ca2+ accumulation is pH-dependent, increasing as the pH is increased above 7.5. Inhibition of Ca2+ accumulation is observed on preincubation with ATP, but not with CTP, UTP, GTP, ADP, adenosine 5'-[beta gamma-methylene]triphosphate or adenosine 5'-[beta gamma-imido]triphosphate. The presence of Ca2+, but not Mg2+, during the preincubation, prevents the effect of ATP + Pi on Ca2+ accumulation. The ATP + Pi inhibition of Ca2+ accumulation is not due to modification of the ATPase catalytic cycle, but rather to stimulation of a rapid Ca2+ efflux from actively or passively loaded vesicles. This Ca2+ efflux is inhibited by dicyclohexylcarbodi-imide. Photoaffinity labelling of sarcoplasmic-reticulum membranes with 8-azido-[alpha-32P]ATP resulted in specific labelling of two proteins, of approx. 160 and 44 kDa. These proteins were labelled in the presence of Pi, but not other anions.     

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.     

Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase

A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.     

Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes.

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5'-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.     

Effects of spin-labeled stearates on duck erythrocyte adenylate cyclase.

The effects of various spin-labeled stearates on duck erythrocyte adenylate cyclase were investigated. Only 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl caused an increase in adenylate cyclase activity. It increased the basal rate by about 50%, and the activities stimulated by isoproterenol and isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate by 35%. Upon analysis of the width parameter delta1 in the electron spin resonance spectra for both the basal enzyme activity and the stimulation obtained with effectors such as guanosine 5'-[beta,gamma-imido]triphosphate, isoproterenol, isoproterenol plus guanosine 5'-[beta,gamma-imido]triphosphate and NaF, a correlation of the changes of modification in adenylate cyclase activities was found. These findings suggest that the molecular environment of the enzyme has been modified.     

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.     

Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.     

Cell surface complexes (''cortices'') isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies.

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5'-[gamma-thio]triphosphate (ATP[S]) and adenosine 5'[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.     

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.     

Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

Beta-agonist-induced inhibitory-guanine-nucleotide-binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma.

In the sarcolemma fraction of foot muscles of a fresh-water bivalve mollusc, Anodonta cygnea, a direct inhibitory, rather than stimulatory, effect of the beta-adrenergic agonist isoproterenol, at micromolar concentration, on cAMP level and adenylate cyclase activity, was revealed. It was blocked by beta- but not alpha-adrenergic antagonists. A single class of [3H]dihydroalprenolol-binding sites with binding properties of beta-adrenergic receptor was detected in mollusc sarcolemma. Potentiation of the inhibitory effect of isoproterenol on mollusc adenylate cyclase activity by GTP or guanosine 5'-[beta,gamma-imido]triphosphate at micromolar concentrations, and its elimination in the presence of guanosine 5'-[beta-thio]diphosphate, were shown. The pertussis-toxin-induced ADP-ribosylation of sarcolemma 40-kDa protein [immunochemically related in the C-terminal part to pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein (G-protein) alpha subunits of vertebrates], as well as the treatment of mollusc sarcolemma with antisera responsive to the C-terminus of vertebrate inhibitory G-protein (G(i)) alpha subunit led to elimination of the inhibitory effect of isoproterenol on adenylate cyclase activity. The results obtained suggest that beta-agonist-induced inhibition of adenylate cyclase in A. cygnea foot muscle may be realized via the beta-adrenoreceptor/G(i) signalling pathway.     

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.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.