Nucleoside Triphosphate Concentrations Research Articles

Sensing growth rate

One of the key elements by which bacteria adjust their growth rate to match nutrient availability in the diverse environments that they inhabit is rRNA synthesis, which directly regulates the rate of ribosome synthesis by a translational feedback mechanism. Gaal et al. now report that growth rate controls rRNA transcription via concentrations of nucleoside triphosphates (NTPs), which are a measure of the nutritional state of the cell. Unlike typical Escherichia coli promoters, the rrn P1 ribosomal RNA promoters have unstable open complexes, which are stabilized by high levels of the initiating NTP. This NTP, which is either ATP or GTP for the seven rRNA operons, binds to the open complex with RNA polymerase, increasing the half-life of the complex and, thus, rRNA transcription. The rate of production of rRNA transcripts is therefore highly dependent upon the cellular concentrations of ATP and GTP and not the growth rate per se. These results explain the previously observed lack of dependence of net rRNA synthesis on gene dosage: increasing the number of rRNA operons in a cell does not result in higher rRNA levels. Instead, transcription of each rRNA operon is proportionately decreased. They also raise the possibility that the transcription of other genes in bacteria known to be under growth control operates by a similar mechanism.Gaal, T. et al. (1997) Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria, Science 278, 2092–2097

Effects of Exhausting Exercise and Catecholamines on K + Balance, Acid-base Status and Blood Respiratory Properties in Carp

The potential role of adrenergic mechanisms in the recovery of potassium balance and acid-base status following 5 min of exhausting exercise was studied in carp. The extracellular metabolic H + load after exercise matched the lactate load, suggesting similar release rates of H + and lactate from white muscle. Blockage of α-adrenoceptors by phentolamine or β-adrenoceptors by propranolol neither influenced absolute magnitudes nor recovery kinetics of extracellular H + and lactate loads. The arterial oxygen tension increased following exercise, but blood oxygen transport was not improved via a red cell β-adrenergic response or modulation of the red cell nucleoside triphosphate content. Exercise induced an increase in extracellular [K +] which was corrected within 30–60 min of recovery. The recovery of K + balance was not influenced by blockage of adrenergic receptors. Red cell [K +] changed only insignificantly following exercise, whereby a possible function of the red cells as a temporary depository for K + during the extracellular hyperkalaemia could not be established. The minimal influence of catecholamines on the measured parameters during recovery from exercise was supported by an absence of change in these parameters upon adrenaline injection in resting carp.

Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria.

The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA (rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis.

Stability of nucleoside triphosphate levels in the red cells of the snake

Nucleated red cells in the nonpregnant garter snake (Thamnophis elegans) contain relatively high concentrations of nucleoside triphosphate (NTP), largely in the form of ATP, which is found at concentrations of approximately 10 mmol l-1 relative to cell volume and 15 mmol l-1 relative to cell water. During pregnancy, levels of NTP in adult red cells rise by approximately 50 % concomitant with an increase in blood progesterone level. The stability of the NTP pool within these red cells was assessed by maintaining cells in vitro at 20 °C, without exogenous nutrients, and in the presence and absence of the metabolic inhibitors iodoacetate and/or cyanide. After 96 h, NTP levels in adult red cells not exposed to the inhibitors had decreased by only approximately 10 %, while in the presence of both inhibitors NTP levels had fallen by less than 50 %. Red cell NTP levels were not affected by acute exposure to high concentrations of progesterone either in vivo or in vitro. NTP levels were much more labile when the cells were maintained in vitro at either low or high pH. Maintenance of red cells at pH 6.0 for 24 h resulted in a decrease in NTP levels of approximately 50 % and at pH 10.0 the levels fell by approximately 80 %, while buffers containing only ATP caused no detectable degradation. Incubation at low or high pH promoted some cell swelling; however, the magnitude of the decreases in intracellular NTP concentration prompted by these pH levels could not be mimicked by incubation of red cells in hypotonic buffer. Total nonspecific ATPase activity at pH 6.0 was approximately 55 % greater than that at pH 7.4, while at pH 10.0 it was approximately 6 % of that at pH 7.4. The pH-dependent decrease in intracellular NTP levels cannot, therefore, be due to stimulation of ATPase activity, at least not at high pH. Overall, the data are consistent with the hypothesis that an appreciable portion of the NTP within these cells is compartmentalized in a stable, but pH-sensitive, pool sequestered from intracellular ATP-hydrolyzing processes.

Hypermutagenic in vitro transcription employing biased NTP pools and manganese cations

In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA polymerase may be rendered hypermutagenic by employing biased nucleoside triphosphate (NTP) concentrations and manganese cations. Using the E. coli R67 plasmid-encoded dihydrofolate reductase (DHFR) gene as target substitution rates approaching 4×10 −2 per base per reaction could be achieved, on a par with hypermutagenic reverse transcription. In all cases the majority of substitutions was that expected from the NTP pool bias. The addition of manganese ions increased the frequency of mutations, particularly the proportion of transversions. Functional DHFR hypermutants with up to 8% amino acid substitutions were readily obtained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.

Effects of hypoxia and hyperoxia on oxygen-transfer properties of the blood of a viviparous snake

Red cell oxygen affinity, red cell nucleoside triphosphate (NTP) levels and blood oxygen-carrying capacity were determined for male, nonpregnant and pregnant female, and fetal garter snakes Thamnophis elegans exposed to hypoxia (5 % oxygen) and hyperoxia (100 % oxygen). Male and nonpregnant female snakes were maintained under these conditions for up to 3 weeks and exhibited an apparent maximal change in oxygen affinity after 14 days of hypoxia and hyperoxia. Red cell NTP levels decreased and oxygen affinity increased with exposure to hypoxia, while exposure to hyperoxia promoted an increase in red cell NTP concentrations and a decrease in red cell oxygen affinity in the males. Hyperoxia-exposed nonpregnant females did not show a significant change in oxygen affinity. After 14 days of hypoxia, the pregnant females showed an increase in red cell oxygen affinity which was associated with a decrease in red cell NTP concentration and in the molar ratio of NTP/hemoglobin relative to normoxic controls. Fourteen days of hyperoxia did not result in a change in oxygen affinity of red cells from the pregnant female, but did promote a slight increase red cell NTP concentrations. The blood parameters of fetuses from females exposed to hypoxia or hyperoxia did not differ from those of normoxic control fetuses. The fetuses of females exposed to hypoxia suffered greater mortality, appeared less developed and had a lower average wet mass than the fetuses of normoxic- and hyperoxic-exposed females. Neither hypoxia nor hyperoxia altered the oxygen-carrying capacity of the blood in any group of snake.

Relative phosphocreatine and nucleoside triphosphate concentrations in cerebral gray and white matter measured in vivo by 31P nuclear magnetic resonance

Rates of ATP metabolism generally are higher in cerebral gray matter compared to white matter. In order to study the physiology of this regional difference in vivo, the 1-dimensional chemical shift imaging technique (1D-CSI) was used to acquire 31P nuclear magnetic resonance spectra from 2.5 mm slices of 4-week old piglet brains. Spectra from predominantly gray matter slices (estimated 76% gray matter, 7 mm below the scalp) were compared to predominantly white matter slices (56% estimated white matter, 13 mm below the scalp) as assessed by magnetic resonance images. The 1D-CSI technique introduced no systematic changes in the ratio of signals from a single chamber phantom containing a phosphocreatine (PCr) and ATP solution. Gray matter slices showed a PCr/NTP ratio of 0.93 ± 0.11 (mean ± S.D.) using a 2 s interpulse interval, a value very close to the ratio in surface coil localized spectra. The predominantly white matter slices showed a PCr/NTP ratio of 1.32 ± 0.18 ( P < 0.02 for gray versus white matter). Using the estimated percentages of gray and white matter in the two slices and calculated concentrations from fully relaxed spectra, the gray matter PCr/NTP ratio is approximately 0.77, while the ratio in white matter is approximately 2.18. The difference in PCr/NTP measured in vivo suggests that either the total NTP concentration is higher or the steady state PCr concentration is lower in gray matter than in white matter in the piglet brain.

The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA.

An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase. It requires the presence of the upstream activation sequence on supercoiled DNA. Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM. Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place. The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration. On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate. These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA. In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.

Transcription against an applied force.

The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.

Quantitative relationship between brain temperature and energy utilization rate measured in vivo using 31P and 1H magnetic resonance spectroscopy.

In neonatal and adult animals, modest reduction in brain temperature (2-3 degrees C) during ischemia and hypoxia-ischemia provides partial or complete neuroprotection. One potential mechanism for this effect is a decrease in brain energy utilization rate with consequent preservation of brain ATP, as occurs with profound hypothermia. To determine the extent to which modest hypothermia is associated with a decrease in brain energy utilization rate, in vivo 31P and 1H magnetic resonance spectroscopy (MRS) was used to measure the rate of change in brain concentration of phosphocreatine, nucleoside triphosphate, and lactate after complete ischemia induced by cardiac arrest in 11 piglets (8-16 d). Pre-ischemia metabolite concentrations and MRS-determined rate constants were used to calculate the initial flux of high energy phosphate equivalents (d[approximately P]/dt, brain energy utilization rate). Baseline physiologic and MRS measurements were obtained at 38.2 degrees C and repeated after brain temperature was adjusted between 28 and 41 degrees C. This was followed by measurement of d[approximately P]/dt during complete ischemia at 1-2 degrees C increments within this temperature range. Adjusting brain temperature did not alter any systemic variable except for heart rate which directly correlated with brain temperature (r = 0.95, p < 0.001). Before ischemia brain temperature inversely correlated with phosphocreatine (r = -0.89, p < 0.001), and reflected changes in the phosphocreatine-ATP equilibrium, because brain temperature inversely correlated with intracellular pH (r = -0.77, p = 0.005). Brain temperature and d[approximately P]/dt were directly correlated and described by a linear relationship (slope = 0.61, intercept = -12, r = 0.92, p < 0.001). A reduction in brain temperature from normothermic values of 38.2 degrees C was associated with a decline in d[approximately P]/dt of 5.3% per 1 degree C, and therefore decreases in d[approximately P]/dt during modest hypothermia represent a potential mechanism contributing to neuroprotection.

Reduction of Cytochrome aa3 Measured by Near-Infrared Spectroscopy Predicts Cerebral Energy Loss in Hypoxic Piglets

Near-infrared spectroscopy is a noninvasive monitoring technique that allows quantitative measurement of changes in cerebral oxygenated Hb (HbO2), deoxygenated Hb (Hb), total Hb, and oxidized cytochrome aa3 (CytO2). Changes in cerebral Hb oxygenation and CytO2 have been measured in human neonates and infants under a variety of conditions. However, the association of these measurements with cerebral high-energy phosphate loss is not known. We studied simultaneous changes in cerebral HbO2, Hb, total Hb, and CytO2 by near-infrared spectroscopy and changes in nucleoside triphosphate (NTP, mostly ATP) and phosphocreatine (PC) concentrations and intracellular pH by in vivo 31P-labeled magnetic resonance spectroscopy. Four-wk-old piglets (n = 8) underwent sequential hypoxic episodes of increasing severity (inspired O2 concentration, 12, 8, 6, 4, and 0%). Animals were anesthetized and mechanically ventilated. At all levels of hypoxia, cerebral HbO2 decreased, and Hb increased. Loss of PC or NTP was not observed until inspired O2 concentration was decreased to less than 12%. With such severe hypoxia, hypotension, intracellular acidosis, and increasingly severe PC and NTP depletions occurred. Decreases in PC and NTP correlated closely with decreased CytO2 and arterial blood pressure (p < 0.0001) but not with changes in HbO2 and Hb. In conclusion, cerebral hypoxemia is readily detected by near-infrared spectroscopy as a decrease in HbO2 and an increase in Hb. However, relative changes in cerebral HbO2 and Hb have low predictive value for cerebral energy failure. Reduction of CytO2 is highly correlated with decreased brain energy state and may indicate impending cellular injury.

Nucleotide Requirements for Activated RNA Polymerase II Open Complex Formation in Vitro

The role of nucleotides in activated RNA polymerase II transcription was studied. Permanganate footprinting confirmed that there is a strict nucleotide requirement for forming open promoter complexes that cannot be overcome by the addition of a dinucleotide primer corresponding to the start site sequence. However, higher concentrations of other nucleoside triphosphates can substitute for ATP in catalyzing open complex formation. Opening catalyzed by these nucleotides is inhibited by the ATP analogue adenosine 5'-O-(thio-triphosphate), suggesting that they may function through cross-binding to the ATP site. The KM for ATP for opening and the involvement of other nucleotides in opening differs from the characteristics reported for TFIIH helicase and C-terminal domain kinase activities. This raises the possibility that opening does not involve these activities. The results alleviate very significantly the considerable current uncertainty concerning the role of ATP in the mammalian mRNA transcription initiation pathway.

Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate.

Transcription terminators recognized by several RNA polymerases include a DNA segment encoding uridine-rich RNA and, for bacterial RNA polymerase, a hairpin loop located immediately upstream. Here, mutationally altered Escherichia coli RNA polymerase enzymes that have different termination efficiencies were used to show that the extent of transcription through the uridine-rich encoding segment is controlled by the substrate concentration of nucleoside triphosphate. This result implies that the rate of elongation determines the probability of transcript release. Moreover, the position of release sites suggests an important spatial relation between the RNA hairpin and the boundary of the terminator.

RAPID AND SIMULTANEOUS MEASUREMENT OF ANODIC AND CATHODIC HAEMOGLOBINS AND ATP AND GTP CONCENTRATIONS IN MINUTE QUANTITIES OF FISH BLOOD

Oxygen transport to vertebrate tissues is dependent upon (a) the intrinsic O2-binding properties of haemoglobin (Hb) and (b) modulation by allosteric effectors, such as nucleoside triphosphates (NTP), that depress Hb O2-affinity in fish red cells. While some species, such as flatfish, primarily use adenosine triphosphate (ATP) to modulate O2-binding to Hb, others, such as eel and carp, also use guanosine triphosphate (GTP) (Kono and Hashimoto, 1977; Leray, 1982; Weber and Jensen, 1988). Unlike mammals, fish exhibit high degrees of molecular and functional Hb heterogeneity, which is manifested interspecifically but is also evident from polymorphism in different individuals of the same species (Weber, 1990). While some species such as carp have multiple Hbs that migrate anodally in normal electrophoresis and exhibit similar intrinsic oxygenation properties and sensitivities to NTP (Gillen and Riggs, 1972), others such as trout and eel have both anodic and cathodic components that exhibit different intrinsic O2-binding properties and different sensitivities to cofactors. Having higher O2 affinities and lower Bohr effects, the cathodic Hbs may be better adapted for O2 transport under hypoxic, hypercapnic or acidotic conditions than the anodic ones from the same species (Hashimoto et al. 1960; Binotti et al. 1971; Weber et al. 1975; Weber, 1990). Ambient hypoxia induces decreases in erythrocytic NTP (Wood and Johansen, 1972), which safeguards blood O2-loading in the gills. The concentrations of the major NTPs and anodic or cathodic Hbs are thus important indices of respiratory and adaptational status in fish. Erythrocytic ATP and GTP levels are generally assayed by thin layer or column chromatography or enzymatically, total Hb is measured by spectrophotometry, and the anodic and cathodic composition of the Hbs present by electrophoresis or isoelectric focusing. We here report a method for rapid and simultaneous assay of ATP and GTP, and of anodic and cathodic Hbs, in minute (approximately 3 &micro;l) quantities of fish blood. We used trout [Oncorhynchus mykiss, 101&plusmn;8 g (s.e.m.); N=9], eel (Anguilla anguilla, 114&plusmn;17 g; N=9) and carp (Cyprinus carpio, 1407&plusmn;104 g; N=9) supplied by local pisciculturists. The fish were kept for at least 1 week in 1 m3 glass fibre tanks with aerated running fresh water at 15 &deg;C before experimentation. Blood samples were drawn from the caudal blood vessels into heparinised syringes.

Pregnancy-Associated Factors Affecting Organic Phosphate Levels and Oxygen Affinity of Garter Snake Red Cells

Pregnancy in Thamnophis elegans is associated with an increase in the nucleoside triphosphate (NTP) concentration and a concomitant decrease in the oxygen affinity of the adult red cell. Red cell NTP levels rise at about the time of ovulation and peak during mid-gestation. Since plasma progesterone levels appear to have a similar profile, we examined the influence of progesterone on this phenomena. Surgical removal of the corpora lutea (CL), primary site of progesterone release, plus fetuses abolished the pregnancy-associated effect on red cell NTP levels. Removal of the CL alone resulted in NTP levels intermediate to those of the red cells of the nonpregnant and the pregnant groups. Progesterone implants in nonpregnant females, as well as in males, caused red cell NTP concentrations to rise. These data support the hypothesis that progesterone, secreted by the CL, in the presence of the fetus, is largely responsible for the pregnancy-associated increase in the red cell NTP concentration.

Decreased energy requirement of toad retina during light adaptation as demonstrated by 31P nuclear magnetic resonance.

1. The effect of light and dark adaptation on the levels of phosphorus metabolites (nucleotide di- and triphosphates, phosphocreatine, pyridine nucleotide, inorganic phosphate, phosphodiesters, phosphomonoesters, and uridine diphosphate-glucose) in the toad (Bufo marinus) retina and retinal extracts was studied by 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Spectra were acquired using an NMR probe specifically designed for superfusion and illumination of a single retina. Retinae were maintained at a steady state for up to 10 h in an electrolyte solution containing 10 mM Hepes buffer and bubbled with 98% O2-2% CO2, pH 7.8 at 20 degrees C. 3. The intracellular concentrations of the phosphorus metabolites were measured in total darkness or during prolonged exposure to light. The concentration of nucleoside triphosphates (NTP) in the dark-adapted retina was about 1.5 mM and that of phosphocreatine (PCr) was about 0.7 mM. 4. In saturating levels of light, 6.0 x 10(11) or 1.5 x 10(13) quanta s-1 cm-2 at 520 nm, the levels of PCr and phosphomonoesters rose, the levels of NTP and protons (pH) were maintained, and the levels of pyridine nucleotides and nucleotide diphosphates (NDP) fell. 5. A rise in the level of PCr in the presence of an unchanged level of NTP in the light-adapted retina indicates that the energy consumption of the retina is greater in the dark. 6. These results are in agreement with the results of oxygen consumption, glucose dependence, and electrophysiological studies which also indicate that the metabolic energy requirement of the retina decreases in light.

Profound, reversible energy loss in the hypoxic immature rat brain

The goal of this study was to compare the effects of oxygen deprivation on cellular energy state and pH in the developing and adult rat brain. Relative quantities of phosphocreatine (PC), inorganic phosphorus (P i), and nucleoside triphosphates (NTP), and intracellular pH, were determined using in vivo 31P NMR spectroscopy at different postnatal ages (postnatal day (P) 2–6, P9–13, P16–20, P23–27) in the hypoxic rat brain (7 min, 4% O 2). While a significant increase in P i was seen at all ages during hypoxia, a severe but reversible reduction in concentrations of PC (80–100% decrease) and NTP (40–50% decrease) was observed only at P9–13. This dramatic response was not seen in older (> P16) or younger (< P6) animals. These latter groups responded with moderate decreases in brain PC (50–60% decrease) and NTP (20–40% decrease). In addition, the youngest animals showed much less intracellular brain acidosis than the other age groups. The transient period of development during which the brain exhibits heightened susceptibility to hypoxic energy failure coincides with known changes in brain energy production pathways and susceptibility to hypoxia-induced excitability.

Arterial Po2, acid‐base status, and red cell nucleoside triphosphates in rainbow trout transferred from fresh water to 20‰ sea water

Transfer of rainbow trout from fresh water to 20‰ sea water decreased dorsal aortic Po2 from 90 to 60 mmHg within 1 h. Arterial Po2 was depressed for 24 h before it returned to control values. The internal hypoxia rapidly reduced the red cell nucleoside triphosphate content, suggesting prompt regulation of blood O2 affinity. The extracellular acid‐base status changed towards a small mixed metabolic and respiratory acidosis.

Arterial PO2, acid-base status, and red cell nucleoside triphosphates in rainbow trout transferred from fresh water to 20% sea water

Transfer of rainbow trout from fresh water to 20‰ sea water decreased dorsal aortic Po2 from 90 to 60 mmHg within 1 h. Arterial Po2 was depressed for 24 h before it returned to control values. The internal hypoxia rapidly reduced the red cell nucleoside triphosphate content, suggesting prompt regulation of blood O2 affinity. The extracellular acid-base status changed towards a small mixed metabolic and respiratory acidosis.

Biochemical bases for difference in oxygen affinity of maternal and fetal red blood cells of rattlesnakes

Pregnancy in Crotalus viridis oreganus is associated with an increase in the nucleoside triphosphate (NTP) concentration and a concomitant decrease in the oxygen affinity of the adult red blood cell. However, although the red blood cells of non-pregnant adults and fetuses have indistinguishable NTP concentrations, they have different oxygen affinities. Therefore, red blood cell NTP concentrations alone cannot account for the oxygen-affinity difference between fetal and maternal red blood cells. Hemoglobins from adult and fetal snakes had similar intrinsic oxygen affinities; however, adult hemoglobin was more responsive to organic phosphate modulation compared with fetal hemoglobin. Structural differences, indicated by native gel electrophoresis and electrophoresis of the globins under denaturing conditions at high pH, corroborated functional differences of hemoglobins from fetus and adult. Therefore, the biochemical basis for the oxygen-affinity difference between maternal and fetal red blood cells in this rattlesnake appears to be unique. It appears to be caused by a functionally distinct fetal hemoglobin and the pregnancy-associated rise in red blood cell NTP levels in the mother.