Occurrence and Ecological Significance of GTP in the Ocean and in Microbial Cells

A comparison between the ATP concentrations based on peak height light emission values (0 to 3 s) and integrated light flux determinations (15 to 75 s) for a variety of seawater samples revealed that the integrated method of light detection consistently yielded higher ATP concentrations, ranging from 1.38 to 2.35 times larger than the corresponding peak ATP values. A significant correlation (r = 0.923) was observed for a plot of ΔADP (i.e., integrated ATP - peak ATP) versus GTP + UTP, suggesting that the analytical interference on the ATP assay was the result of the presence of non-adenine nucleotide triphosphates. Size-fractionation studies revealed an enrichment of the non-adenine nucleotide triphosphates, relative to ATP, in the smallest size fraction analyzed (<10 μm). Investigations were conducted with 20 species of unicellular marine algae to determine their intracellular nucleotide concentrations, and these determinations were compared to the levels measured in lab cultures of the marine bacterium Serratia marinorubra. These results indicated that the intracellular GTP/ATP ratios in S. marinorubra increase in direct proportion to the rate of cell growth, and that the GTP/ATP ratios in bacteria are much greater than in growing algae, presumably due to the differences in rates of cellular biosynthesis. It is concluded that quantitative determinations of GTP/ATP ratios in environmental sample extracts may be useful for measuring microbial growth.     

cGMP influences guanine nucleotide binding to frog photoreceptor G-protein

A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light-induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light-induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors.     

Uridylation of U6 RNA in a nuclear extract in Ehrlich ascites tumor cells

The uridylation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells was examined. This reaction required ATP or GTP, although these nucleotides were not incorporated into U6 RNA itself. ATP and GTP could be replaced by their nonhydrolyzable analogues ATP gamma S and GTP gamma S. Therefore, hydrolysis of ATP or GTP is not necessary for the uridylation of U6 RNA, indicating that these nucleotides are effectors of this reaction. By chromatographies of a nuclear extract of Ehrlich ascites tumor cells on phosphocellulose and DEAE-cellulose, U6 RNA could be separated from an enzyme adding a uridine residue(s) to this RNA.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

A method to determine 18 O kinetic isotope effects in the hydrolysis of nucleotide triphosphates

A method to determine 18 O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of 18 O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18 O at sites of mechanistic interest also contains 13C at all carbon positions, whereas the 16 O-labeled nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1(333)-catalyzed hydrolysis of [beta18 O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 microg nucleotide reaction product.     

Circadian spontaneous bioluminescent glow and flashing ofGonyaulax polyedra

Summary A new, fully computerized method for the measurement and analysis of dinoflagellate bioluminescence has been developed and applied to the spontaneous light emission ofGonyaulax polyedra. This light emission consists of a low-level steady glow, and occasional superimposed flashes. The instrumentation distinguishes the two components and records them separately; both exhibit circadian rhythmicity. In this paper we describe the method in detail, and show results for flashing and glow measured under light: dark cycles and under constant light of different intensities. Under constant dim light at 19°C, both rhythms exhibit two peaks during a circadian cycle; the minor ones occur approximately nine hours before the major ones. Under these conditions the major flashing peak occurs early during the subjective night, and the major glow peak at the end, about nine hours later. However, the relative phase angle between glow and flashing peaks varies with light intensity, being as little as 220 min (3.7 h) in the dark under light-dark entrained conditions, to as much as 700 min (11.7 h) in dim light under free-running conditions. The ambient light intensity also affects differentially the amount of light emitted in the two modes of spontaneous luminescence. These results suggest that the controls for the two processes must at some point diverge.     

Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s(-1)) and is accelerated by eEF1Balpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Balpha binding to eEF1A (10(7)-10(8) M (-1) s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.     

Guanosine and formycin triphosphates bind at non-catalytic nucleotide binding sites of CF1 ATPase and inhibit ATP hydrolysis

Guanosine triphosphate and formycin triphosphate (FTP) in the presence of excess Mg²⁺ can bind to empty non-catalytic sites of spinach chloroplast ATPase (CF₁). This results in a greatly reduced capacity for ATP hydrolysis compared to the enzyme with non-catalytic sites filled with ATP. With two GTP bound at non-catalytic sites the inhibition is about 90%; with two FTP bound about 80% inhibition is obtained. Binding and release of the nucleotides from the non-catalytic sites are relatively slow processes. Exposure of CF₁ with one or two empty non-catalytic sites to 5–10 μM FTP or GTP for 15 min suffices for about 50% of the maximum inhibition. Reactivation of CF₁ after exposure to higher FTP or GTP concentrations requires long exposure to 2 μM EDTA. The findings show that, contrary to previous assumptions, GTP can bind tightly to non-catalytic sites of CF₁. They suggest that the presence of adenine nucleotides at non-catalytic sites might be essential for high catalytic capacity of the F₁ ATPases.     

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase.     

Multiple inhibitory and activating effects of nucleotides and magnesium on adrenal adenylate cyclase.

Adenylate cyclase in particulate fractions from rat adrenal glands is subject to regulation by purine nucleotides, particularly guanine nucleotides. While GTP activates the enzyme, this effect is not evident in all particulate fractions. Following dialysis of the refractory fractions activation by GTP is observed, an indication that endogenous nucleotides may obscure the effects of added GTP. The analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p gives considerable more activity than does GTP. GDP, on the other hand, is inhibitory, an effect revealed only in the absence of a nucleotide-regenerating solution. GDP blocks the action of both GTP and Gpp(NH)p. These results show that the gamma-phosphate of the nucleotide is required for but need not be metabolized in the activation process. At low substrate concentration (0.1 mM ATP or adenyl-5'-yl imidodiphosphate) stimulation of the enzyme by ACTH occurs only in the presence of added guanine nucleotide (GTP or Gpp(NH)p); the hormone and nucleotide act synergistically. While both GTP and Gpp(NH)p inhibit fluoride-stimulated activity, the level of fluoride required to demonstrate such inhibition appears not to be related to the level of fluoride required for activation of the enzyme. In the presence of GTP, or GTP plus ACTH, the enzyme exhibits normal Michaelis-Menten kinetics with respect to substrate utilization (K-m equal to 0.16 mM). In the activated state, produced with ACTH plus GTP, the enzyme is less susceptible to inhibition by a species of ATP uncomplexed with Mg2+, but is more susceptible to inhibition by Mg2+. These results demonstrate that fundamental differences exist between different states of the adenylate cyclase. The difficulties in describing kinetically the regulation of adenylate cyclase systems in view of the multiple actions of nucleotides and magnesium are discussed.     

Surface receptor-mediated activation of adenylate cyclase in Dictyostelium. Regulation by guanine nucleotides in wild-type cells and aggregation deficient mutants

GTP and GTP analogs produced significant (up to 17-fold) and persistent activation of adenylate cyclase in lysates of Dictyostelium discoideum amoeba. The activation was enhanced 2- to 4-fold by cAMP (the agonist for receptor-mediated adenylate cyclase activation), was specific for guanine nucleoside triphosphates, and was inhibited by guanosine 5'-(O-2-thio)diphosphate. The order of potency of guanine nucleotides was guanosine 5'-(O-3-thio)triphosphate greater than guanyl-5'-yl imidodiphosphate greater than GTP; half-maximal activation was observed with 1-10 microM guanine nucleotide. Maximal activation occurred when the guanine nucleotide was added within seconds after cell lysis and the lysate was preincubated for 5 min prior to assay. Under these optimal in vitro conditions, the capacity of guanine nucleotides to activate decreased, closely correlating with adaptation or desensitization induced by exposure of intact cells to cAMP during a period of 10 min. These data strongly support that regulation of adenylate cyclase in Dictyostelium occurs via a receptor-linked GTP/GDP exchange protein. Two mutants, designated synag 7 and 49 were isolated in which cAMP and/or guanine nucleotides were not sufficient to activate adenylate cyclase. The wild-type pattern of guanine nucleotide regulation was restored to synag 7 lysates by the addition of a high-speed supernatant from wild-type cells. Characterization of these mutants demonstrates that activation of adenylate cyclase is not required for growth or cell-type specific differentiation but is essential for cellular aggregation and influences morphogenesis and pattern formation. This suggests that Dictyostelium may provide a model suitable for detailed genetic analysis of surface receptor-guanine nucleotide-binding regulatory protein linked adenylate cyclase systems and for determining the role of these systems in development.     

Binding of periodate-oxidized guanine nucleotides to eukaryotic elongation factor 2

Periodate-oxidized guanine nucleotides (GTPox and GDPox) were shown to bind stoichiometrically to rat liver elongation factor 2 (EF-2). This binding was quantitatively inhibited in the presence of GTP. After binding, oxidized nucleotides remained on EF-2 despite extensive dialysis. They exchanged, however, with free quanine nucleotides in the course of prolonged (greater than 1 h) incubations. The prior reduction EF-2.GTPox with NaBH4 abolished, to a large extent, this slow exchange. Thus, a Schiff's base was implicated to be formed between EF-2 and oxidized guanine nucleotides. Mg2+ increased the GTPox concentration necessary for a stoichiometric binding to EF-2. EF-2-oxidized nucleotide conjugates bound in the presence of ribosomes a second molecule of GTP (or GTPox). GTPox bound to EF-2 in the presence of ribosomes appeared to exchange readily with free GTP. Moreover, GTPox proved to be active as substrate in EF-2 and ribosome-dependent GTPase reaction: Km values found for GTPox and GTP were 7.7 and 3.4 microM, respectively. The binding of GTPox to EF-2 inhibited only partially the subsequent ribosome-dependent GTP binding, and GTPase reaction or polyphenylalanine (polyPhe) synthesis. On the other hand, the binding of GuoPP[CH2]Pox to EF-2 inhibited all of these reactions strongly. The nature of the binding site involved in the direct interactions of EF-2 with guanine nucleotides is discussed in the light of these results.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

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.     

Transient response of retinal rod outer segment phosphodiesterase to actinic light pulses. I. Simple quantitative kinetic model

We present a quantitative kinetic model for the transient velocity (microM of cGMP hydrolyzed/s) response of retinal rod outer segment (ROS) cGMP phosphodiesterase (v(t) versus t) to a stimulating light pulse in the linear response range. The model gives an excellent fit to experimental v(t) versus t data for ROS suspensions at different concentrations of GTP and GDP and clarifies experimental results which are difficult to understand in the absence of such a model. It contains the minimum number of steps required to fit our experimental data and consists of one rate-limiting step with specific rate kL for the production of active phosphodiesterase (PDE), PDE*, by photoactivated rhodopsin, R*, and deactivation processes for R* and PDE* with lifetimes tau R and tau P, respectively. The experimental graphs of v(t) versus t at each concentration of GTP and GDP are characterized by a fast rise to a peak value, vpeak, followed by a slow decay to zero level. The minimal kinetic model allows us to characterized completely the effects of GTP and GDP, and any other pertinent species, in terms of their effects on the parameters kL, tau R, and tau P. Our kinetic model indicates that for "washed" ROS preparations (a) the risetime of v(t) is determined by tau P which has a value of about 2 s and is insensitive to [GTP]. (b) The decay of v(t) is determined by tau R which decreases with [GTP] and has a value greater than 300 s at low [GTP] and a limiting value of 50 s at high [GTP]. We attribute the greater than 300 s lifetime to the complex R*G (where G is ROS G protein) and the 50-s lifetime to free R*. (c) The rate kL increases hyperbolically with [GTP] with a half-maximal value of 56 microM and kL.max = 22-45 s-1. (d) Peak velocity is given by the expression vpeak alpha kL tau P which is consistent with the dependence of kL on [GTP] and the experimental finding that vpeak varies hyperbolically with [GTP]. The minimal model has also allowed us to (a) develop clear definitions of amplification for the light-triggered enzymatic cascade and (b) clarify experimental methods for measuring gain.(ABSTRACT TRUNCATED AT 400 WORDS)     

The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides

The mechanism of interaction of the G-protein of retinal rods with rhodopsin and with nucleotides has been investigated using two independent techniques, light-scattering and direct binding measurements with labeled nucleotides. Binding of photoexcited rhodopsin (R*) and nucleotides are shown to be antagonist, and three conformations of the G-protein are described, each of which is proposed to be related to a different level of light-scattering, as follows: (a) the "dark" state, stable in the absence of photoexcited rhodopsin, in which the nucleotide site is poorly accessible and has a high affinity (dissociation constants, 0.1 microM for GDP and 0.01 microM for GppNHp); (b) the R*-bound state in which the nucleotide site is rapidly accessible with a lower affinity (dissociation constants, about 20 microM for GDP and GTP; 20-100 microM for GppNHp). Binding of R* to the G-protein therefore enables rapid binding or exchange of the nucleotide; this in turn reduces the affinity of the G-protein for R* (dissociation constants, 0.2 microM for G-protein with GDP bound and 2-10 microM for G-protein with GppNHp bound, compared to 1 nM in absence of bound nucleotide); and (c) the third state, the activator of the phosphodiesterase. In the presence of GTP, an additional irreversible and fast step, which is proposed to be the dissociation of alpha-GTP from beta gamma, is shown to occur; a steady state equilibrium is obtained, and the dissociation constant measured between GTP and this third state of the G-protein in the presence of R* is an apparent constant which depends on the rate of transconformation between the first two states and on the rate of GTP hydrolysis. The minimum value of this apparent dissociation constant for GTP (0.05-0.1 (microM) is obtained at high levels of illumination. Finally, some results (number of nucleotide sites and saturation of the rate of the light-scattering signal) suggest an oligomeric association of the G-protein.     

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.     

Systematic variations in the content of the purine nucleotides in the steady-state perfused rat heart. Evidence for the existence of controlled storage and release of adenine nucleotides

1. The contents of the major purine nucleotides in the isolated non-working perfused rat heart varied systematically during 80min of perfusion. In particular the amounts of ATP, ADP, GTP, cyclic AMP and cyclic GMP in the well-oxygenated myocardium showed changes ranging from 25 to 60% of the mean concentrations. The apparent periodicity was about 30min for some and about 60min for other nucleotides. 2. These data are in contrast with measurements of parameters reflecting heart performance, which remained constant over this period of perfusion. 3. The ATP/ADP ratio, the cyclic AMP content, the GTP content and the GTP/GDP ratio in the tissue bore a constant relationship to one another, and all showed the same temporal variation. 4. Increasing the energy demand on the heart by administration of bovine somatotropin (1μg/ml) tended to damp the variations, and generally lower the content of all the nucleotides. 5. The total extractable adenine nucleotide pool also showed systematic temporal variations of as much as 1.3μmol/g wet wt. of tissue within 10min. 6. These variations could not be accounted for as inter-conversion with adenosine, other purine nucleotides, nucleosides or purine-degradation products either in the tissue or in the perfusion medium. No evidence was found in this preparation of the purine nucleotide oscillations described by Lowenstein and his co-workers [see Tornheim & Lowenstein (1975) J. Biol. Chem. 250, 6304–6314]. 7. Further, the pool size increases cannot be satisfactorily explained by either synthesis de novo or the breakdown of any purine macromolecular species in the cell. Thus it is suggested that an unsuspected substantial storage form of purine nucleotide may exist in heart.     

Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.     

Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol

Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.