Hydrolysis of ribonucleoside 3'-diphosphates by rye grass 3'-nucleotidase.

Rye grass 3'-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2'-O-methyladenosine 3'-monophohate and 2'-deoxyadenosine 3'-monophosphate 35.8 and 542 times more slowly than the normal substrate (3'-AMP), verifying the importance of the 2'-beta-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3'-AMP, both the 2'-O-methyl and 2'-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3'-AMP (K-m equals 0.12 mM), with apparent K-i's of 0.39 and 0.51 nM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3'-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3'-phosphohydrolase activities, the 3'-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3'-diphosphate and guanosine tetraphosphate. Neither adenosine 3'-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes

Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation. We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways. In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI). On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate. At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.     

Activation of ppGpp-3'-pyrophosphohydrolase by a supernatant factor and ATP

The breakdown of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) into GDP and PPi is catalyzed by a Mn2+-dependent 3'-pyrophosphohydrolase, the translation product of the spoT gene. The escherichia coli enzyme is normally found to be associated with the "crude" ribosome fraction. It is reported here that the guanosine 5'-diphosphate, 3'-diphosphate 3'-pyrophosphohydrolase activity in this fraction is activated by ATP in the presence of a relatively heat-stable, low molecular weight, supernatant factor (BS100). This stimulation is not due to a removal of reaction products such as by the phosphorylation of GDP to GTP or by the hydrolysis of PPi. Hydrolysis of ATP is probably required because neither adenosine 5'-(3-thio)triphosphate nor adenosine 5'-(beta, gamma-imido)triphosphate can substitute for ATP. Levallorphan, a morphine analog, which had been shown to inhibit in vivo ppGpp degradation, inhibits specifically the stimulation of ppGpp hydrolysis by ATP and the supernatant factor. The possible relationship of this system and the in vivo energy-dependent control of ppGpp degradation is discussed.     

Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase. Purification and substrate specificity

The regulatory nucleotide guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and its precursor guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) are accumulated during stringent response in bacterial cells. The enzyme pppGpp-5'-phosphohydrolase, which catalyzes the conversion of pppGpp to ppGpp, was partially purified from Escherichia coli. It has Mr = 140,000 and an apparent Km of 0.11 mM for pppGpp. It requires Mg2+ and a monovalent cation. NH4+ is preferred over K+, while Na+ is inactive. The enzyme does not hydrolyze GTP, ATP, pppApp, or ppGpp. It is also not effectively inhibited by these nucleotides. pppGpp-5'-phosphohydrolase hydrolyzes the 3'-monophosphate analog pppGp equally well (apparent Km of 0.13 mM), yielding the recently identified MS III nucleotide (ppGp). pppGpp-5'-phosphohydrolase does not have RNA 5'-terminal gamma-phosphatase activity; however, 5'-terminal phosphates are released by pppGpp-5'-phosphohydrolase when the GTP-terminated RNA chains are first converted into oligonucleotides by RNase A treatment. pppGpp-5'-phosphohydrolase was found to actively hydrolyze the dinucleotide fragment pppGpNp but exhibited very low activity toward longer chain fragments. The 3'-unphosphorylated dinucleotide pppGpN was, however, not hydrolyzed. The ability of pppGpp-5'-phosphohydrolase to hydrolyze pppGpp, pppGp, and pppGpNp, but not pppG and pppGpN, indicates that pppGpp-5'-phosphohydrolase is rather nonspecific toward the 3'-OH substitutions of the substrates although a free, unsubstituted phosphate group at the 3'-OH position is essential.     

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.     

Degradation of guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT gene product of Escherichia coli.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) is rapidly degraded to guanosine 5'-diphosphate (ppG) and probably pyrophosphate by an enzyme present in the ribosomal fraction prepared from spoT+ strains of Escherichia coli. The ppGpp-degrading enzyme was released from the ribosomes during dissociation at low ionic strength. Ribosomes are not essential for degradation of ppGpp, and decay of ppGpp is strictly dependent on manganese ions. The reaction is sensitive to inhibition by tetracycline, which can be reversed by MnCl2, indicating that the inhibitory effect is due to the previously described chelating properties of the antibiotic. When the ppGpp-degrading enzyme was complemented with adenosine 5'-triphosphate (pppA) and a nucleoside diphosphate kinase, decay of ppGpp was accelerated yielding pppG and ppG as major products. In the absence of pppA we have been unable to detect the ppGpp-degrading enzyme in various spoT- mutant strains indicating that this enzyme is the spoT gene product.     

A laser Raman spectroscopic study of the interaction of the methylmercury cation with AMP, ADP and ATP

The interaction of the CH3Hg+ cation with adenosine 5'-monophosphate, adenosine 5'-diphosphate, and adenosine 5'-triphosphate has been studied in aqueous solution at neutral pH by laser Raman spectroscopy. Metal binding is shown to occur preferentially at the N-1 ring position of adenine, with some indication of coordination to the N-7 site and substitution of a proton on the exocyclic NH2 group of the nucleic base. Binding of the cation to phosphate groups also occurs extensively, with both the -PO2-3 and -PO-2 groups. The equilibrium constants for the binding to the phosphate groups and for N-1 coordination are approx. 70 and 600 M-1, respectively.     

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.     

Fermentative Accumulation of Guanosine Polyphosphate by Brevibacterium ammoniagenes

Guanosine-3'-diphosphate-5'-monophosphate (3.35 mg/ml), guanosine-3'-diphosphate-5'-diphosphate (MSI) (5.21 mg/ml), and guanosine-3'-diphosphate-5'-triphosphate (MSII) (0.82 mg/ml), in addition to guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate, were accumulated by microbial conversion of 5'-xanthylic acid with a mutant of Brevibacterium ammoniagenes.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.     

Nitrofurantoin prompts the stringent response in Bacillus subtilis

Nitrofurantoin causes the stringent response in Bacillus subtilis. After exposure of a stringent strain to this drug, the intracellular concentrations of guanosine 3'-diphosphate 5'-diphosphate (ppGpp), guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and ATP increased, while that of GTP decreased. In a relaxed strain no accumulation of ppGpp or pppGpp was observed, but both GTP and ATP declined after the addition of nitrofurantoin. Protein synthesis was equally sensitive to nitrofurantoin in both the stringent and relaxed strains, but the drug inhibited RNA accumulation only in the stringent strain, not in the relaxed strain. Nitrofurantoin also caused the accumulation of ppGpp in Escherichia coli and Serratia marcescens.     

Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response.

We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.     

Mutation spoT of Escherichia coli increases expression of the histidine operon deleted for the attenuator.

F'-episomes carrying the Salmonella typhimurium wild-type or attenuator-deleted histidine (his) operons were introduced into Escherichia coli strains containing relA or spoT single and double mutations known to affect guanosine 3'-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'-triphosphate 5'-diphosphate (pppGpp) levels. Expression of the his operon and expression of the gene for 6-phosphogluconate dehydrogenase (gnd) were measured during balanced growth in amino acid-rich and minimal media. The data were consistent with the interpretation that ppGpp is a positive effector of his operon expression, whereas pppGpp is not an essential effector. The conclusion that his operon expression is maximally stimulated at a lower than maximum intracellular ppGpp concentration was further confirmed. Neither ppGpp nor pppGpp appeared to influence gnd gene expression. The metabolic regulation of the E. coli his operon was found to be similar to the ppGpp-meidated metabolic regulation of the S. typhimurium his operon.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Effect of guanosine tetraphosphate on in vitro protein synthesis directed by E1 and E3 colicinogenic factors.

The in vitro synthesis of colicin E3 was found to be stimulated by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), while that of several other ColE3 plasmid-specific proteins was reduced in the presence of this nucleotide. The ColE1 plasmid-directed in vitro synthesis of colicin E1 was also found to be stimulated by ppGpp.     

Characterization of the guanosine 5'-triphosphate 3'-diphosphate and guanosine 5'-diphosphate 3'-diphosphate degradation reaction catalyzed by a specific pyrophosphorylase from Escherichia coli.

Guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) are specifically degraded by a manganese-dependent pyrophosphorylase present in spoT+ but not in spoT- strains of Escherichia coli, indicating that the enzyme is the spoT gene product. The enzyme catalyzes the release of pyrophosphate from the 3' position of ppGpp or pppGpp, yielding ppG and pppG, respectively; pppGpp could not be detected as an intermediate in the decay reaction. Degradation of (p)ppGpp is optimal in the presence of 200 to 300 mM potassium or sodium acetate, at a pH of 7.5 to 8 and a temperature of 37 degrees C.     

Elevation of guanosine 3',5'-monophosphate level by adenosine in cerebellar slices of guinea pig.

Effect of adenosine on the level of guanosine 3',5'-monophosphate in guinea pig cerebellar slices was investigated. Adenosine increased the concentration of guanosine 3',5'-monophosphate in the slices 3--4 fold. Upon removal of adenosine from the medium, the concentration of guanosine 3',5'-monophosphate returned to the initial level. AMP, ADP or ATP also increased the guanosine 3',5'-monophosphate level to the same extent as adenosine, while adenine or other nucleosides were not effective. In the absence of Ca2+ in the incubation medium, adenosine did not increase the concentration of guanosine 3',5'-monophosphate in cerebellar slices although level of adenosine 3',5'-monophosphate was elevated by adenosine. Anticholinergic agents, adrenergic blocking agents or antihistaminics did not prevent the increase of guanosine 3',5'-monophosphate by adenosine indicating that the effect of adenosine was not mediated by the release of neurotransmitters. The combination of adenosine with depolarizing agents showed an additive effect on the level of guanosine 3',5'-monophosphate indicating that adenosine increased the level of guanosine 3',5'-monophosphate by a different mechanism from the depolarization.     

Partial purification and characterization of cytidine 5''-diphosphate-diglyceride hydrolase from membranes of Escherichia coli.

Cytidine 5'-diphosphate (CDP)-diglyceride is hydrolyzed to phosphatidic acid and cytidine 5'-monophosphate by a specific membrane-bound enzyme in cell-free extracts of Escherichia coli. The hydrolase can be extracted from the particulate fraction with Triton X-100 and purified 1,000-fold in the presence of this detergent. Several nucleoside disphosphate diglycerides were synthesized to determine the substrate specificity of the hydrolase. CDP-diglyceride was hydrolyzed preferentially, although uridine 5'-diphosphate-diglyceride, guanosine 5'-diphosphate-diglyceride, and adenosine 5'-diphosphate (ADP)-diglyceride were also slowly hydrolyzed. Surprisingly, the purified enzyme did not catalyze detectable cleavage of deoxy-CDP (dCDP)-diglyceride. The liponucleotide pool of E. coli contains dCDP-diglyceride and CDP-diglyceride in approximately equal amounts (Raetz and Kennedy, 1973). Water-soluble nucleoside pyrophosphates, such as CDP-choline, nicotinamide adenine dinucleotide, or adenosine 5'-triphosphate are not attacked by this specific hydrolase. Hydrolysis of CDP-diglyceride is strongly inhibited by adenosine 5'-monophosphate and by ADP-diglyceride.