Effect of wounding on nucleotide pools inBidens pilosa L.

Wounding both cotyledons ofBidens pilosa (var.radiatus) induces the inhibition of hypocotyl growth. The wound signal is transmitted very rapidly from cotyledon to hypocotyl and can be visualized by the change in nucleotide pools. First we have shown that the irradiance of the plant can change the ATP level without plant wounding. Therefore, plants were harvested at the start of the light period. Under these conditions, we have determined in hypocotyl the levels of adenosine triphosphate (ATP), guanosine triphosphate (GTP) and non adenylic triphosphates (NTP), and adenylate energy charge (AEC) after wounding. We have observed a transient (2 min) increase in the ATP level followed by a decrease 5 to 30 min later. A similar result was obtained for the GTP level but with some delay. The GTP level increased in 5 min and then decreased after 60 min. For the NTP level the decrease is effective from 5 to 60 min after wounding. The calculation of AEC has shown that a very tight control in the level of ATP may be involved in response to wounding.     

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 nucleotides 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.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

The adenosine triphosphate pool and the adenyl cyclase activity inEscherichia coli strains with different UV sensitivity

Two genetically relatedEscherichia coli strains were tested for the ATP pool and for the adenyl cyclase activity in the cell membrane fractions. TheEscherichia coli strain B_S1with a high UV sensitivity showed a higher ATP level than the wild typeEscherichia coli B strain. The adenyl cyclase activity was found to be lower in the sensitive strain than in the wild typeEscherichia coli.     

The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.     

High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system

The overexpression of subunit b of F₁F₀ adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 μg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F₁F₀ ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.     

High-yielding cascade enzymatic synthesis of 5-methyluridine using a novel combination of nucleoside phosphorylases

Abstract

A novel combination of Bacillus halodurans purine nucleoside phosphorylase (BhPNP1) and Escherichia coli uridine phosphorylase (EcUP) has been applied to a dual-enzyme, sequential, biocatalytic one-pot synthesis of 5-methyluridine from guanosine and thymine. A 5-methyluridine yield of >79% on guanosine was achieved in a reaction slurry at a 53 mM (1.5% w/w) guanosine concentration. 5-Methyluridine is an intermediate in synthetic routes to thymidine and the antiretroviral drugs zidovudine and stavudine.     

Evidence that ATP exerts control of the rate of glucose utilization in the intact Escherichia coli cell by altering the cellular level of glucose-6-P, an intermediate known to inhibit glucose transport in vitro

The effects of treating nitrogen-starved cultures of $\text{Escherichia coli}$ W4597 (K) with various doses of 2,4-dinitrophenol include increases in the rates of glucose utilization, decreases in ATP and glucose-6-P and maintenance of the level of fructose-1, 6-P₂. A quantitative correlation was observed between the increases in the rates of glucose utilization and decreases in glucose-6-P in agreement with the observation made $\text{in vitro}$ that glucose-6-P inhibits glucose transport in $\text{E. coli}$. A quantitative correlation was also observed between glucose-6-P and ATP indicating that the fall in glucose-6-P is effected by the fall in ATP which indirectly signals increased glucose utilization and increased ATP production.     

Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l⁻¹ with a yield of 1.02 g g⁻¹ total sugars during anaerobic fermentation.     

Antimutagenic effects of spermine and guanosine in continuous cultures of Escherichia coli mutator strain mutH.

Summary In E. coli strain RH531 containing mutator gene mutH ^–, whose mechanism of enhanced mutability depends upon DNA replication, spermine and guanosine reduce the rates of mutation to T5^R and to increased fitness.     

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli

The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized by Escherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F₁‐part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth‐arrested cells, which is vital for its use in two‐stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW^?1 h^?1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth‐coupled processes) as well as volumetric productivity (in two‐stage processes) depending on which of the performance measures is more crucial for the process and product of interest.     

Reazioni Metaboliche All'auxina - VII Variazioni a Carico Di Metaboliti Della Glicolisi

Abstract

Metabolic responses to auxin. VII. Effects on glycolysis. — The detection of changes the of level of respiratory substrates could provide an indication on the site of action of auxin. The effects of indoleacetic acid on the contents in several glycolysis intermediates in isolated segments from the growing part of the third internode of etiolated pea seadlings were thus investigated.

Hexose phosphates, triose phosphates, phospho-enol-pyruvate, pyruvate and ethanol were determined enzymatically in the neutralized perchloric extracts from groups of segments incubated for 3 hours in the presence or in the absence of 5 × 10 ^?5 M indoleacetic acid. No significant difference appeared between the results on the perchloric extracts and those obtained after fractionation with barium and alcohol, according to Umbreit et al.

Acetaldehyde was measured as by Klotz. Pyruvate was determined also as a phenyl-hydrazone, according to Cavallini and Frontali; the same method was used for the determination of α-ketoglutarare and of an unknown compound, showing a chromatographic behavior similar to that of an uronic acid.

Auxin induced a modest but significant increase of hexose-6-phosphates (16%) and of fructose di-phosphate (38%). For glucose-1-phosphate and triose phosphates — present at very low concentration — no significant changes could be detected, α-ketoglutarate level appeared unchanged. The compound behaving as an uronic acid increased, in the auxin treated segments, by more than 100%.

A very definite response to auxin appeared at the level of pyruvate. acetaldehyde and alcohol. Pyruvate quite reproducibly increased by 60%, and acetaldehyde production was more than doubled. In contrast, alcohol decreased by about 40%.

These results are discussed in relation to the previous data on the effects of auxin on the oxidative metabolism, and, particularly, to the findings of an increased rate of CO-sensitive respiration and of the rise of the ATP, TPNH and red. glutathione levels in the auxin treated pea internode segments.     

Relation of ATP and creatine phosphate to fast axoplasmic transport in mammalian nerve

—ATP and creatine phosphate (CP) levels in cat sciatic nerve maintained in vitro were measured. Anoxia produced by N₂ or NaCN or the uncoupling of phosphorylation with DNP reduced the combined levels of ATP + CP to approximately one-half of control levels within 15 min. These agents also blocked fast axoplasmic transport in vitro within 15 min. A block of glycolysis with iodoacetic acid (IAA) reduced the combined levels of ATP + CP to approximately one half of control levels within 1.5–2 h and exposure of nerve in vitro to IAA caused a block of fast axoplasmic transport within the same interval. The correlation of the time at which block of transport occurred with the fall in the level of high-energy phosphates is consistent with the hypothesis that ATP supplies the energy required by the mechanism underlying fast exoplasmic transport.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis

Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis. Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined. The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis. An unexpectedly high cross-reactivity was revealed between the EF-Tu of B. subtilis and the antiserum against the EF-Tu of E. coli. A comparison of the predicted amino acid sequences from the tuf-genes of E. coli and B. subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.Abbreviations EF-Tu elongation factor Tu - GDP guanosine 5-diphosphate - GTP guanosine 5-triphosphate - MCF microcomplement fixation - T type strain     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.