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PubMed2002

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthetic activities on Escherichia coli SpoT domains.

Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5'-diphosphate 3'-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, alpha-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further alpha-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA -, spoT- double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).

Blotting, Western↗
PubMed1975

Regulation of RNA synthesis in Escherichia coli. III. Degradation of guanosine 5'-diphosphate 3'-diphosphate in cold-shocked cells.

Cold-shocked cells of Escherichia coli can degrade intracellularly accumulated guanosine 5'-diphosphate 3'-diphosphate (ppGpp). The rate of ppGpp degradation is governed, as in whole cells, by the spoT gene; a rapid breakdown reaction is associated with the presence of the spoT+ allele and at least a five-fold slower decay occurs in spoT-minus mutants. The two degradation reactions in shocked cells display the following similarities: (i) the rates of degradation are equivalent to whole cell estimates, (ii) both require a full complement of activated amino acids, (iii) both are dependent upon supplements in the reaction mixture which stimulate the availability of energy-rich compounds and (iv) neither is inhibited by concentrations of ribosomal antibiotics which severely restrict protein synthesis. Apart from characteristic rate differences, decay of ppGpp in shocked cells derived from spoT-minus strains is discerned from spoT+ mediated decay in shocked cells by sensitivity to high concentrations of tetracycline and by manganese ion dependence.

Alleles↗
PubMed1975

The involvement of guanosine 5-diphosphate-3-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli. Lack of ppGpp inhibition of acyltransfer from acyl-ACP to sn-glycerol 3-phosphate.

The response of the Escherichia coli sn-glycerol-3-phosphate acyltransferase to guanosine 5-diphosphate-3-diphosphate (ppGpp) has been determined in vitro employing palmityl coenzyme A (CoA) and palmityl acyl carrier protein as acyl substrates. Levels of ppGpp which cause significant inhibition of enzyme activity with palmityl-CoA as substrate have no effect on enzyme activity when palmityl acyl carrier protein is employed as acyl donor. The inhibition of enzyme activity observed with palmityl-CoA as acyl substrate was dependent upon the relative concentrations of MgCl2 and ppGpp (MgCl2 to ppGpp ratio) employed. With palmityl-CoA as acyl donor, PPGpp inhibited the production of lysophosphatidic acid but not phosphatidic acid. With palmityl acyl carrier protein as acyl substrate, ppGpp had no influence upon the distribution of the reaction products.

Acyltransferases↗
PubMed1979

Role of the spoT gene product and manganese ion in the metabolism of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli.

Addition of divalent ion chelating agents picolinic acid, 1,10-phenanthroline, or quinoline-2-carboxylic acid to wild type, relA, or relX, but not spoT strains of Escherichia coli increases the levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Poorly chelating analogs of these agents and a larger and more highly charged chelating agent, ethylene glycol bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid are ineffective. Mn2+ reverses the increase in ppGpp. The increase in ppGpp in wild type cells can be explained by an inhibition of degradation. In spoT cells the response is more complex; ppGpp does not increase although degradation is completely inhibited. The lack of increase in spoT cells suggests a role for spoT in synthesis of ppGpp in addition to its known role in degradation. Growth of both spoT+ and spoT cells is inhibited following chelator addition. This suggests that growth inhibition is through a mechanism not directly involving ppGpp. The results of this study provide evidence in intact cells for a role for Mn2+ and the spoT gene product in ppGpp degradation, and provide further evidence for an involvement of spoT and possibly divalent ions in ppGpp synthesis.

Alleles↗
PubMed1981

Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli.

Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P. A., and Chang, F. N. (1980) J. Bacteriol. 144, 499-508). The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation. Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount. We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined. It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants. The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance. It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.

Arginine↗
PubMed1994

Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate.

The three-dimensional structures of complexes of yeast apotransketolase with the coenzyme analogs 6'-methyl, N1'-pyridyl, and N3'-pyridyl thiamin diphosphate, respectively, were determined with protein crystallographic methods. All three coenzyme analogs bind to the enzyme in a fashion highly similar to the cofactor thiamin diphosphate. Thus, either one of the hydrogen bonds of the pyrimidine ring nitrogens to the protein is sufficient for proper binding and positioning of the cofactor. The lack of catalytic activity of the N3'-pyridyl analog is not due to incorrect orientation of the pyrimidine ring, but results from the absence of the hydrogen bond between the N1' nitrogen atom and the conserved residue Glu418. The structure analysis provides further evidence for the importance of this conserved interaction for enzymatic thiamin catalysis.

Coenzymes↗
PubMed1994

Adenosine 5'-diphosphate binding and the active site of nucleoside diphosphate kinase.

The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+. The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle. The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence. The adenine base makes only nonpolar contacts with the protein. It points outside, explaining the lack of specificity of NDP kinase toward the base. The ribose 2'- and 3'-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains. A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side. The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion.

Adenosine Diphosphate↗
PubMed1985

Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose).

Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP-ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA.

Animals↗
PubMed1982

Interaction of 3'-O-(1-naphthoyl)adenosine 5'-diphosphate, a fluorescent adenosine 5'-diphosphate analogue, with the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier protein in the mitochondrial membrane.

3'-O-(1-Naphthoyl)adenosine 5'-diphosphate (N-ADP), a fluorescent analogue of ADP, was established as a potent inhibitor of adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) transport in mitochondria and inside-out sonic particles; the Ki value was about 5 microM. The inhibition was of a mixed type. On the other hand, N-ADP was not transported in a measurable way in either type of particles. Upon binding to the particles, the fluorescent intensity of N-ADP was decreased; the release of the bound N-ADP upon addition of carboxyatractyloside (CATR) to mitochondria and bongkrekic acid (BA) to sonic particles was reflected by increases of fluorescence. In parallel assays dealing with 14C-labeled N-ADP, specifically bound [14C]N-ADP was equated to [14C]N-ADP released upon addition of either CATR (mitochondria) or BA (sonic particles). The specific binding of N-ADP corresponded to 1.4-1.6 nmol/mg of protein in mitochondria, with a Kd value of 3 microM, and to 1.5-1.6 nmol/mg of protein in sonic particles, with a Kd value of 6 microM. Essentially similar values were obtained for N-ATP binding. These values are at least twice as high as those found for specific ADP or ATP binding, suggesting that N-ADP or N-ATP binds to potential nucleotide binding sites that were not totally occupied by ADP or ATP. Whereas nearly all the specifically bound N-ADP in mitochondria was displaced by an excess of ADP (400 microM) at pH 7.4, only 30% could be removed from sonic particles under the same conditions. Furthermore at pH 6.5, no more than half of the specifically bound N-ADP could be removed by excess ADP in mitochondria and only 10-20% in sonic particles. These results indicate that each ADP/ATP carrier unit contains at least two types of nucleotide sites capable of interacting with N-ADP. Because of the hydrophobic nature of the naphthoyl moiety of N-ADP, the data suggest that difference in N-ADP binding in mitochondria and sonic particles are related to differences in the hydrophobic nature of their sites. Due to the special features of N-ADP (strong specific binding to the ADP/ATP carrier and no competence for transport), this DP analogue was particularly suitable for investigating the sensitivity of the nucleotide binding sites of the carrier to chemical modifiers. Inactivation studies were therefore carried out with mitochondria and sonic particles to compare the sensitivity to UV light and butanedione of the binding of N-ADP, [3H]BA, and [14C]Ac-CATR, a radiolabeled substitute for CATR. Both in mitochondria and in sonic particles, UV light and butanedione inactivated more rapidly the binding of N-ADP than that of [3H]BA. On the other hand, in mitochondria, UV light inactivated more rapidly the binding of [14C]Ac-CATR than that of N-ADP; the reverse was true for the inactivation by butanedione. The inactivation data conclusively indicate that BA, CATR, and adenine nucleotides are recognized by different specific sets of amino acids.

Adenosine Diphosphate↗
PubMed1987

Facile synthesis of 2-[(3-aminopropyl)thio]adenosine 5'-diphosphate: a key intermediate for the synthesis of molecular probes of adenosine 5'-diphosphate function.

Adenosine 5'-diphosphate (ADP) and, uniquely, its C-2 derivatized analogues are able to induce platelet activation. We here report the synthesis of 2-[(3-aminopropyl)thio]-ADP from ADP itself via 1,N6-etheno-ADP. 2-[(3-Aminopropyl)thio]-ADP induced platelet aggregation with a potency about one-seventh that of ADP itself and should prove a useful intermediate in the synthesis of other probes of platelet function.

Adenosine Diphosphate↗
PubMed1985

Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins.

Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule-associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co-purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP.

Animals↗
PubMedDate not supplied

HPLC analysis of cyclic adenosine diphosphate ribose and adenosine diphosphate ribose: determination of NAD+ metabolites in hippocampal membranes.

Cyclic adenosine diphosphate-ribose (cADPR) and ADPR were separated by high-performance liquid chromatography (HPLC) on a CarboPac PA-1 column at strong basic pH and quantitated by a pulsed amperometric detector. Although this HPLC method was quite sensitive and highly reproducible, it did not allow the separation of cADPR from guanosine monophosphate (GMP) which, when present, could be removed by ion-affinity chromatography, using gel-immobilized Fe3+ columns. Crude synaptic membranes from rat hippocampi were incubated with nicotinamide adenine dinucleotide (NAD) and acidic extracts were subject to HPLC analysis after neutralization. Incubation led to a time-dependent formation of ADPR, which was amplified when membranes were incubated in the presence of guanosine trisphosphate (GTP), guanosine-5'-0-(3-thiotrisphosphate) (GTP-gamma-S) or AlF3. cADPR did not accumulate in detectable amounts and only a minimal proportion (< 5%) of radioactivity originating from [3H]NAD co-eluted with authentic cADPR in extracts from hippocampal membranes. The simultaneous detection of cADPR and ADPR we have described may help the search for inhibitors of cADPR metabolism, which will allow to measure the cADPR that accumulates under basal conditions or in response to extracellular signals.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

[On the nature of free radicals of adenosine diphosphate in redox system NADH-riboflavin-adenosine diphosphate].

Free radicals formed in redox-system NADH + riboflavin (Rf) containing adenosine diphosphate (ADP) in aqueous solutions at room temperature were investigated by ESR-method. Besides formation during the reaction of semireduced form of ADP heterocycle which is identified by singlet ESR signal arising with halfwidth of about 20 Gs the correlations were shown to appear between intensities of weak low-field and high-field lines, symmetrical relatively field values corresponding to g-factor near 2.00. This was interpreted as the evidence of free radicals formation: PO(3)2-. and free radicals of ADP phosphate fragment.

Adenosine Diphosphate↗
PubMed1975

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.

Alcohol Oxidoreductases↗
PubMed1978

Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

Exogenous addition of guanosine and adenosine 5'-(mono, di and tri) phosphate 3'-diphosphates (pppGpp, ppGpp, pGpp, pppApp, ppApp and pApp) stimulated the synthesis of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli. From the results obtained with ppGpp and pppApp, this effect appeared to be at a transcriptional level and depended greatly on the growth condition; the largest effect was observed in cells under shiftdown or grown on poor enrgy source. ppGpp and pppApp, unlike cyclic AMP, did not act to overcome the inhibition of enzyme induction by glucose, but in combination with cyclic AMP caused a synergistic stimulation effect. In the shiftdown cells, ppGpp and pppApp gave 30% or more stimulation effect on tryptophanase induction while cyclic AMP did not stimulate induction. There was therefore a pronounced difference between cyclic AMP and ppGpp or pppApp in stimulatory function.

Adenine Nucleotides↗
PubMed1984

Increase of intraerythrocytic fructose-1,6-diphosphate after incubation of whole human blood with fructose-1,6-diphosphate.

The incubation of whole blood with fructose-1,6-diphosphate (FDP) entails a statistically significant increase of intraerythrocytic FDP together with a decrease of blood glucose. The increase is not significant when equimolar amounts of fructose plus twice molar phosphate are used. The effect of FDP is decreased in the presence of an excess of oxygen. FDP added to the whole blood is removed from plasma by the activity of plasma enzymes and by the presence of blood cells as well. No specific interaction of FDP with plasma proteins seems to occur and the effects of FDP addition last longer than is compatible with the presence of FDP in the plasma.

Adolescent↗
PubMed1983

Microtubule assembly with the guanosine 5'-diphosphate analogue 2',3'-dideoxyguanosine 5'-diphosphate.

The GDP analogue 2',2'-dideoxyguanosine 5'-diphosphate (ddGDP) supports efficient tubulin polymerization. Microtubule-associated protein (MAP) dependent microtubule assembly occurs in 0.1 M 2-(N-morpholino)-ethanesulfonate, and sheets of parallel protofilaments are formed in 1.0 M glutamate without MAPs. The nucleotide is bound to tubulin in the course of polymerization, presumably in the exchangeable GTP site. The ddGDP is not hydrolyzed, however, and is completely stable in the reaction mixture. Nor was the nonexchangeable GTP bound to tubulin hydrolyzed in ddGDP-supported polymerization: equivalent amounts of GTP remained associated with polymerized tubulin after polymerization with either ddGDP or GTP. Higher concentrations of ddGDP than GTP were required under all conditions. Nevertheless, under optimum conditions for the ddGDP-supported reaction, polymerization began with a shorter lag period and both the rate and extent of polymerization were greater with ddGDP than with GTP. The MAP-dependent reaction with ddGDP is temperature dependent, cold reversible, and inhibited by calcium and antimitotic drugs. It differs from the GTP-supported reaction in being most vigorous at minimal Mg2+ concentrations and exquisitely sensitive to GDP inhibition.

Animals↗