guanosine-monophosphate and 5--guanylylmethylenebisphosphonate

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.

Topics: Cell Membrane; Cholera Toxin; Cyclic GMP; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Leukotriene B4; Neutrophils; Pertussis Toxin; Receptors, Immunologic; Receptors, Leukotriene B4; Virulence Factors, Bordetella

Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation.

Topics: Animals; Cattle; Diphosphonates; Glycerol; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Tubulin

Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cattle; Caudate Nucleus; Diphosphonates; Enzyme Activation; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; In Vitro Techniques; Magnesium

The different functional complexes of ribosomes with elongation factor F (EF-G) were studied by digestion experiments with trypsin. It was found that upon interaction of EF-G with ribosomes the L7/L12 proteins are sensitive to trypsin and are trypsin resistant after dissociation of EF-G from ribosomes. The significance of conformational alterations in the L7/L12 and also in the other proteins in the translation process is discussed.

Topics: Bacterial Proteins; Diphosphonates; Escherichia coli; Fusidic Acid; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Peptide Elongation Factor G; Peptide Elongation Factors; Protein Conformation; Ribosomal Proteins; Ribosomes; Trypsin

The effect of certain antiviral compounds said to act because of an increased permeability of virally infected cells has been tested in SFV-infected BHK cells. The time at which SFV-infected BHK cells become sensitive to the action of GppCH2p is more than an hour later than the time at which protein synthesis in such cells becomes depressed. The uptake of [3H]GppCH2p is the same in infected and uninfected cells, whether measured at 19 or 37 degrees C. We conclude that GppCH2p, and probably other 'impermeant' inhibitors of protein synthesis also, affect virally-infected cells selectively not because of an increased permeability, but because of a general impairment of protein synthesis in such cells.

Topics: Animals; Antiviral Agents; Cell Line; Cell Membrane Permeability; Cricetinae; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Kidney; Protein Biosynthesis; Semliki forest virus; Togaviridae Infections; Virus Diseases

tRNAPhe and tRNAVal of Escherichia coli were derivatized at the S4U8 position with p-azidophenacyl and p-azidophenacylacetate photoaffinity probes. The modified tRNAs could still function efficiently in all of the partial reactions of protein synthesis except for an approximately sevenfold decrease in the rate of translocation. Irradiation (310 to 340 nm) of probe-modified Phe-tRNA or Val-tRNA placed in the ribosomal A site led to crosslinking that was totally dependent on irradiation, the presence of the azido group on the probe, mRNA, and elongation factor Tu (EFTu). Prephotolysis of the modified tRNA abolished crosslinking, but prephotolysis of the ribosomes and factors had little effect. Crosslinking was efficiently quenched by mercaptoethanol or dithiothreitol, demonstrating accessibility of the probe to solvent. Use of GDPCP in place of GTP also blocked crosslinking, probably because of the retention of EFTu on the ribosome. Crosslinking with the p-azidophenacyl acetate (12 A) probe was only half as efficient as with the p-azidophenacyl (9 A) probe, and this ratio was not changed by varying Mg2+ from 5 to 15 mM. The crosslink was from a functional A site, since AcPhePhe-tRNA at the A site could be crosslinked, and it was A site-specific, because neither translocation nor direct non-enzymatic P site binding yielded any significant covalent product. The crosslink was to ribosomal protein(s) of the 30 S subunit. No other ribosomal component was crosslinked. Identification of the protein crosslinked is described in the accompanying paper.

Topics: Affinity Labels; Azides; Binding Sites; Diphosphonates; Escherichia coli; Guanosine Monophosphate; Guanosine Triphosphate; Kinetics; Magnesium; Mercaptoethanol; Peptide Elongation Factor Tu; Peptide Elongation Factors; Photolysis; Ribosomes; RNA, Transfer, Amino Acyl; Thiouridine

Addition of 10 microM guanyl-5'-ylimidodiphosphate at 30 degrees or 0 degree to guinea pig brain particulates instantaneously evoked nearly 50% inhibition of adenylate cyclase activity as determined after removal of the GTP analog by washing of the particulates. The inhibitory state, once formed, persisted for at least 60 min as long as the preparation was kept either in a medium devoid of the analog (0-30 degrees) or in its presence at 0 degree. During incubation at 30 degrees in the presence of the analog, however, the inhibited or nontreated enzyme showed a gradual increase in enzyme activity. Both the inhibitory and the activating effects of the analog were saturable, with a half-maximal concentration of about 1.0 microM, and were antagonized by simultaneous addition of GTP, GDP, and GMP (in decreasing order). The persistently inhibited enzyme enabled the detection of marked stimulation by norepinephrine and histamine, whereas these amines showed only marginal stimulation of the enzyme before treatment with the analog. Formation of such a persistent inhibitory state appears to be specific to brain cyclase.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Imidodiphosphate; Animals; Brain; Diphosphonates; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Guinea Pigs; Histamine; Magnesium; Male; Manganese; Norepinephrine; Thionucleotides

The uptake of the GTP analogue guanylyl(beta,gamma-methylene)diphosphonate (GppCH2p) is the same in Semliki Forest virus (SFV)-infected BHK cells as in mock-infected cells, in spite of the fact that protein synthesis is inhibited by GppCH2p more markedly in SFV-infected cells than in control cells. A possible explanation for this difference is that infected cells have a lower concentration of GTP and a lower ratio of GTP:GDP than uninfected cells, and the analogue may thus be a more effective competitive inhibitor of translation. We conclude that in this system, the difference between infected and uninfected cells lies not at the plasma membrane but within the cytoplasm.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cricetinae; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Methylglucosides; Protein Biosynthesis; Semliki forest virus

Topics: Adenylyl Cyclases; Animals; Diphosphonates; Enzyme Activation; Female; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Muscle, Smooth; Rats; Thionucleotides

D-Ala2-Met5-enkephalin, morphine, and noradrenaline inhibit the adenylate cyclase in homogenates of neuroblastoma x glioma hybrid cells in a dose-dependent manner even after the enzyme has been preactivated by cholera toxin. Half-maximal inhibition and extent of inhibition are the same with native or cholera toxin-activated enzyme. The inhibition caused by opioids or noradrenaline are antagonized by naloxone or phentolamine, respectively. The effect of D-Ala2-Met5-enkephalin on cholera toxin-activated enzyme is immediate in onset and rapidly reversed by the addition of naloxone. Guanyl-5'-yl-imidodiphosphate stimulates basal activity but inhibits the enzyme activated by cholera toxin or prostaglandin E1. Stimulation occurs at a concentration of 100 microM or above, inhibition even at 0.1 microM. The inhibitory effect of the non-hydrolysable GTP analog is antagonized by GTP. Guanyl-5'-yl-methylenediphosphonate, another nonhydrolysable GTP analog, inhibits basal as well as cholera toxin-stimulated or prostaglandin E1-stimulated adenylate cyclase. Other guanine derivatives such as GDP, GMP, cyclic GMP, guanyl-5'-yl-phosphoric acid amide and guanosine have no effect under the same conditions. The results may be taken as a piece of evidence for two separate guanyl nucleotide-binding sites accompanying the adenylate cyclase in the hybrid cells and mediating, respectively, stimulation and inhibition of the enzyme by hormones.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Clone Cells; Diphosphonates; Endorphins; Enkephalin, Methionine; Enkephalins; Glioma; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hybrid Cells; Kinetics; Mice; Morphine; Neuroblastoma; Norepinephrine; Rats

Topics: Animals; Cell Membrane Permeability; Complement System Proteins; Cytarabine; Diphosphonates; Erythrocyte Membrane; Guanosine Monophosphate; Guanosine Triphosphate; Lymphoma; Mice; Osmolar Concentration; Rabbits; Sucrose

The infection of animal cells by encephalomyocarditis (EMC) virus lead to a drastic change in membrane permeability towards low mol. wt. compounds. Addition of the nucleotide analogue GppCH2p to the culture medium resulted in a specific inhibition of protein synthesis in EMC virus-infected 3T6 cells. This inhibition was not observed when GTP or ATP were present nor in control mock-infected 3T6 cells. The induction of membrane leakiness after viral infection was not specific for 3T6 cells, as it was also detected in mouse L cells, hamster BHK-21 cells and monkey CV1 cells. The inhibitory action produced by GppCH2p in virus-infected cells was fully reversed upon addition of fresh medium. Moreover, analysis of the proteins synthesized after medium replacement showed a preferential synthesis of cellular proteins. The presence of zinc ions resulted in an inhibition of the cleavage of large viral polypeptide precursors to mature viral proteins. Under these conditions, membrane leakiness as measured by GppCH2p, was not observed. However, this seems to be an effect of zinc ions themselves on the membrane, because inhibition of mature protein formation by other means, such as the presence of amino acid analogues, did not prevent inhibition of translation by GppCH2p in virus-infected cells. Addition of the cap analogues 7mGppp and 2'-O'-mGppp, resulted in specific stimulation of viral protein synthesis in EMC virus-infected 3T6 cells. On the other hand, the presence of 7mGp had no effect on translation. We propose that a specific capping of viral mRNA takes place in the presence of these compounds, and leads to increased stability and greater efficiency in the translation of viral mRNA.

Topics: Diphosphonates; Encephalomyocarditis virus; Guanosine Monophosphate; Guanosine Triphosphate; Protein Biosynthesis; RNA Cap Analogs; RNA Caps

Topics: Animals; Brain; Brain Chemistry; Diphosphonates; Guanine Nucleotides; Guanosine Monophosphate; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Microscopy, Electron; Microtubules; Phosphates; Swine; Tubulin

We have examined the properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate (GMPP(NH)P), and 5'-guanylyl methylenediphosphate (GMPP(CH2)P) to identify features of the assembly or disassembly reactions uniquely related to hydrolysis. The assembly of microtubules with GTP or GMPP(NH)P was similar in terms of rates and extents of assembly, the length distributions, and podophyllotoxin-induced depolymerization. The greater rapidity of GMPP(CH2)P-supported assembly, however, resulted in shorter, more numerous microtubules and the rate of podophyllotoxin-induced depolymerization was consistent with an increased number of concentration of microtubules. Experiments with GTP or analogue incorporation and release indicated that GTP-tubule turnover corresponded to a rate of about 8% of the microtubule protein taken up or released per h. With GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of label uptake by unlabeled microtubules were considerably lower than observed with guanosine triphosphate. We suggest that exchange experiments can reflect contributions from head-to-tail polymerization and polymer length redistribution, but it is not as yet possible to evaluate the relative contributions of each process.

Topics: Animals; Brain; Cattle; Diphosphonates; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Macromolecular Substances; Microtubules; Podophyllotoxin; Tubulin