Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Mitochondrial gene expression in saccharomyces cerevisiae. I. Optimal conditions for protein synthesis in isolated mitochondria

An in vitro mitochondrial protein-synthesizing system, which makes use of intact yeast mitochondria, has been developed in order to study mitochondrial gene expression and its control by nuclear-coded proteins. Studies with this system have revealed that: isolated mitochondria synthesize polypeptide gene products which can be radiolabeled to high specific radioactivities when incubated in a "protein-synthesizing medium" that has been optimized with respect to each of its components; two energy-generating systems, endogenous oxidative phosphorylation and an exogenous ATP-regenerating system, support the highest level of protein synthesis; and the omission of an oxidizable substrate results in the synthesis of two new polypeptides (19.5 and 18 kDa) and a decrease in the amounts of cytochrome c oxidase subunits I and II which are synthesized. They have also revealed that added adenine and guanine nucleotides increase the overall level of protein synthesis and that the added guanine nucleotides facilitate polypeptide chain elongation. Although isolated mitochondria which have been optimized for protein synthesis synthesize normal gene products (McKee, E. E., McEwen, J. E., and Poyton, R. O., (1984) J. Biol. Chem. 259, 9332-9338) they still respond to an added dialyzed S-100 fraction from yeast cells by increasing their level of protein synthesis. This stimulation is observed in the presence of optimal concentrations of GTP, making it unlikely that guanyl nucleotides or enzymes which synthesize them are the sole stimulatory factors present in cellular cytosolic fractions, as suggested by Ohashi and Schatz (Ohashi, A., and Schatz, G. (1980) J. Biol. Chem. 255, 7740-7745).     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.     

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate)

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

Stimulation of rhodopsin phosphorylation by guanine nucleotides in rod outer segments

Porcine rod outer segment (ROS) proteins were phosphorylated in the presence of [gamma-32P]ATP and Mg2+, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by autoradiography. The phosphorylation of rhodopsin, the major protein-staining band (Mr approximately 34 000-38 000), was markedly and specifically increased by exposure of rod outer segments to light; various guanine nucleotides (10 microM) including GMP, GDP, and GTP also specifically increased rhodopsin phosphorylation (up to 5-fold). Adenine nucleotides (cyclic AMP, AMP, and ADP at 10 microM) and 8-bromo-GMP (10 microM) or cyclic 8-bromo-GMP (10 microM) had no detectable stimulatory effect on rhodopsin phosphorylation. GTP increased the phosphorylation of rhodopsin at concentrations as low as 100 nM, and guanosine 5'-(beta, gamma-imidotriphosphate), a relatively stable analogue of GTP, was nearly as effective as GTP. Maximal stimulation of rhodopsin phosphorylation by GTP was observed at 2 microM. GMP and GDP were less potent than GTP. Both cyclic GMP and GMP were converted to GTP during the time period of the protein phosphorylation reaction, suggestive of a GTP-specific effect. Transphosphorylation of guanine nucleotides by [32P]ATP and subsequent utilization of [32P]GTP as a more effective substrate were ruled out as an explanation for the guanine nucleotide stimulation. With increasing concentrations of ROS proteins, the phosphorylation of rhodopsin was nonlinear, whereas in the presence of GTP (2 microM) linear increases in rhodopsin phosphorylation as a function of added ROS protein were observed. These results suggest that GTP stimulates the phosphorylation of rhodopsin by ATP and that a GTP-sensitive inhibitor (or regulator) of rhodopsin phosphorylation may be present in ROS.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

GTP in the mitochondrial matrix plays a crucial role in organellar iron homoeostasis

Mitochondria are the major site of cellular iron utilization for the synthesis of essential cofactors such as iron-sulfur clusters and haem. In the present study, we provide evidence that GTP in the mitochondrial matrix is involved in organellar iron homoeostasis. A mutant of yeast Saccharomyces cerevisiae lacking the mitochondrial GTP/GDP carrier protein (Ggc1p) exhibits decreased levels of matrix GTP and increased levels of matrix GDP [Vozza, Blanco, Palmieri and Palmieri (2004) J. Biol. Chem. 279, 20850-20857]. This mutant (previously called yhm1) also manifests high cellular iron uptake and tremendous iron accumulation within mitochondria [Lesuisse, Lyver, Knight and Dancis (2004) Biochem. J. 378, 599-607]. The reason for these two very different phenotypic defects of the same yeast mutant has so far remained elusive. We show that in vivo targeting of a human nucleoside diphosphate kinase (Nm23-H4), which converts ATP into GTP, to the matrix of ggc1 mutants restores normal iron regulation. Thus the role of Ggc1p in iron metabolism is mediated by effects on GTP/GDP levels in the mitochondrial matrix.     

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins.

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.     

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.     

Induction of Bacterial Differentiation by Adenine- and Adenosine-Analogs and Inhibitors of Nucleic Acid Synthesis

Abstract

Several adenine- or adenosine-analogs, which inhibited growth and decreased the intracellular GTP pool, induced sporulation of Bacillus subtilis. The inducers were added to cultures growing in a medium containing excess ammonium ions, glucose, and phosphate in which cells normally cannot differentiate. They included compounds that are modified in the ribose unit (decoyinine, psicofuranine, cordycepin) or are substituted within the purine ring or at the 6-N position of adenosine (6-methylaminopurine, zeatin, 6-anilinopurine, formycin). Their effects on the cellular concentration of nucleotides were also measured. All sporulation inducers except formycin-A caused a decrease of GMP, GDP and GTP, some by inhibiting IMP dehydrogenase and others by inhibiting GMP synthetase. In contrast, formycin-A caused an increase of GMP while GDP and GTP decreased. Therefore, the compound (signal) controlling sporulation is GDP or GTP but not GMP. Antibiotics inhibiting growth by direct inhibition of nucleic acid synthesis did not induce sporulation.     

Interactions between the subunits of transducin and cyclic GMP phosphodiesterase in Rana catesbiana rod photoreceptors

In bullfrog (Rana catesbiana) rods the activity of cyclic GMP (cGMP) phosphodiesterase was stimulated 10 times by washing disc membranes with an isotonic, GTP-containing buffer. This stimulation was maintained following hydrolysis of GTP and after removal of guanine nucleotides. At least 60-70% of the inhibitory gamma subunit of cGMP phosphodiesterase (P gamma) was physically released from membranes by these washing procedures. When cGMP phosphodiesterase was activated by a hydrolysis-resistant GTP analogue, P gamma was found in the supernatant complexed with the transducin alpha subunit (T alpha) using three chromatography systems. When GTP was used to activate cGMP phosphodiesterase, P gamma was also found in the supernatant complexed with GDP.T alpha. This complex was also isolated using the same three chromatography systems, indicating that P gamma remained tightly bound to T alpha even after bound GTP was hydrolyzed. Interaction with the beta,gamma subunits of transducin, which remained associated with disc membranes, was required for the release of P gamma from the GDP.T alpha complex, which resulted in the deactivation of active cGMP phosphodiesterase. We conclude that during activation of cGMP phosphodiesterase, P gamma is complexed with T alpha (both GTP and GDP forms) in the supernatant and that, following GTP hydrolysis, beta,gamma subunits of transducin are necessary for the release of P gamma from the complex and the resulting inactivation of cGMP phosphodiesterase in frog photoreceptors.     

Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides

Binding of thyrotropin-releasing hormone (TRH) to specific receptors on membranes isolated from GH4C1 pituitary cells was inhibited by monovalent cations and guanyl nucleotides. NaCl and LiCl inhibited TRH binding by 70%, with half-maximal inhibition at 30 mM; RbCl and KCl inhibited only 10% at concentrations up to 150 mM. NaCl decreased both the apparent number and the affinity of TRH receptors and increased the rate of dissociation of TRH from both membrane and Triton X-100-solubilized receptors. Guanyl nucleotides inhibited TRH binding up to 80%, with guanyl-5'-yl imidodiphosphate (Gpp(NH)p) approximately GTP much greater than GDP approximately ATP greater than GMP. GTP and Gpp(NH)p exerted half-maximal effects at 0.3 microM and decreased receptor affinity to one-third of control but did not change receptor number. Gpp(NH)p accelerated the dissociation of TRH from membranes but not from solubilized receptors. The effects of NaCl were independent of temperature, while GTP and Gpp(NH)p were much more inhibitory at 22 degrees C (70%) than at 0 degrees C (10%). Inhibition by NaCl could be reversed by washing the membranes, and inhibition by GTP was reversed if membranes were chilled to 0 degrees C. The inhibitory effects of low concentrations of NaCl and Gpp(NH)p were additive. Neither monovalent cations nor GTP prevented the TRH-receptor complex from undergoing transformation from a state with rapid dissociation kinetics to a slower dissociating form. The results suggest that sodium ion regulates TRH binding by interacting with a site on the receptor, while guanyl nucleotides regulate TRH binding indirectly.     

Nuclear apoptosis induced by isolated mitochondria

We isolated and purified mitochondria from mouse livers and spinach leaves.When added into egg extracts of Xenopus laevis,they caused nuclei of mouse liver to undergo apoptotic changes.Chromatin condensation,margination and DNA ladder were observed.After incubating isolated mitochondria in some hypotonic solutions,and centrifuging these mixtures at mgh speed,we got mitochondrial supernatants.It was found that in the absence of cytosolic factor,the supernatant alone was able to induce apoptotic changes in nuclei.The effective components were partly of protein.DNA fragmentation was partly inhibited by caspase inhibitors AC-DEVD-CHO and AC-YVAD-CHO.Meanwhile,caspase inhibitors fully blocked chromatin condensation.Primary characterization of the nuclear endonuclease(s) induced by mitochondrial supernatants was also conducted.It was found that this endonuclease is different from endonuclease G,cytochrome c-induced nuclease,or Ca^2 -activated endonuclease.     

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

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

Inhibition of GABAB Receptor Binding by Guanyl Nucleotides

Abstract: GTP and GDP decreased the saturable binding of [³H]baclofen or [³H]γ-aminobutyric acid ([³H]GABA) to GABA_B but not GABA_A receptors whereas GMP displayed negligible activity. This effect was specific to guanyl nucleotides and was not mimicked by high concentrations of ATP. The inhibition of ligand binding was the result of a diminished receptor affinity with no change in receptor number. The use of a complete physiological saline solution rather than Tris buffer plus Ca²⁺ or Mg²⁺ increased the potency of GTP at the GABA_B receptor. The results are discussed in relation to the effects of GABA and GTP on adenylate cyclase activity in the brain.     

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m⁷GTP or m⁷GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m⁷GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m⁷GMP, in the presence of AdoMet and GTP, because radioactivity from both [α-³²P]GTP and [³H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m⁷GTP, m⁷GDP, et²m⁷GMP, and m²et⁷GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.     

The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.     

Protein synthesis in Artemia salina

It is thought that eucaryotic elongation factor eEF-Ts catalyzes the replacement of GDP for GTP on eucaryotic elongation factor eEF-Tu. We have found that eEF-Ts displays a strong nucleoside diphosphate phosphotransferase activity. This transferase activity resides in a dimer molecule of a subunit molecular weight close to 30,000. The transfosforylating activity of eEF-Ts results in a stimulatory effect of ATP, GTP, UTP and CTP on protein synthesis provided that GDP is present. The specificity for guanine nucleotides in protein synthesis resides only in eEF-Tu.