Mutational analysis of Arabidopsis thaliana ABCE2 identifies important motifs for its RNA silencing suppressor function

• ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.
• Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.
• Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.
• Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling.

     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

• 1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured.
• 2. The oxygen affinity of fetal blood is higher than that of adult blood.
• 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each.
• 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect.
• 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes.
• 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

     

Characterization and Elucidation of Coordination Requirements of Adenine Nucleotides Complexes with Fe(II) Ions

Abstract

In spite of the significant role of iron ions-nucleotide complexes in living cells, these complexes have been studied only to a limited extent. Therefore, we fully characterized the ATP:Fe(II) complex including stoichiometry, geometry, stability constants, and dependence of Fe(II)-coordination on pH. A 1:1 stoichiometry was established for the ATP:Fe(II) complex based on volumetric titrations, UV and SEM/EDX measurements. The coordination sites of ferrous ions in the complex with ATP, established by ¹H-, ³¹P-, and ¹⁵N-NMR, involve the adenine N7 as well as Pα, Pβ, and Pγ. Coordination sites remain the same within the pH range of 3.1–8.3. By applying fluorescence monitored Fe(II)-titration, we established a log K value of 5.13 for the Fe(ATP)^2? complex, and 2.31 for the Fe(HATP)^? complex. Ferrous complexes of ADP^3? and AMP^2? were less stable (log K 4.43 and 1.68, respectively). The proposed major structure for the Fe(ATP)^2? complex is the ‘open’ structure. In the minor ‘closed’ structure N7 nitrogen is probably coordinated with Fe(II) through a bridging water molecule. The electronic and stereochemical requirements for Fe(II)-coordination with ATP^4? were probed using a series of modified-phosphate or modified-adenine ATP analogues. We concluded that: Fe(II) coordinates solely with the phosphate-oxygen atom, and not with sulfur, amine, or borane in the cases of phosphate-modified analogues of ATP; a high electron density on N7 and an anti conformation of the adenine-nucleotide are required for enhanced stability of ATP analogues:Fe(II) complexes as compared to ATP complexes (up to more than 100-fold); there are no stereochemical preferences for Fe(II)-coordination with either Rp or Sp isomers of ATP-α-S or ATP-α-BH₃ analogues.     

Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l^–1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.Additional problems were encountered in using published cellular carbon: ATP ratios for conversion of ATP data to biomass as carbon. Ratios of POC: ATP in cultures of sheathed blue-green algae reached 550 : i, while non-sheathed forms yielded ratios near values previously reported for plankton communities. Difficulties in applying a uniform conversion factor may be expected in plankton communities containing significant volumes of sheathed blue-greens.     

Multiomics Reveals Ectopic ATP Synthase Blockade Induces Cancer Cell Death via a lncRNA-mediated Phospho-signaling Network

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Highlights

• •Ectopic ATP synthase as a therapeutic target for gefitinib-resistant NSCLC.
• •Multiomics uncovers the dynamic network in response to ecto-ATP synthase blockade.
• •Ecto-ATP synthase blockade induces cytotoxicity by CK2/phospho-topo IIα/GAS5 axis.
• •A positive feedforward circuit between phospho-topo IIα and lncRNA-GAS5.

     

The Decay of the ATPase Activity of Light Plus Thiol-Activated Thylakoid Membranes in the Dark

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the γ subunit of the ATP synthase in the light enhances the rate of Mg²⁺-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH₄Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.     

VARIABILITY IN RATIOS OF PHYTOPLANKTON CARBON AND RNA TO ATP AND CHLOROPHYLL A IN BATCH AND CONTINUOUS CULTURES1,2

The feasibility of estimating phytoplankton carbon and RNA concentrations from measurements of ATP and chlorophyll a (chl a) concentrations was studied using chemostat populations of the marine diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle (= T. fluviatilis Hustedt). C:ATP and RNA:ATP ratios were studied for six additional marine species in batch culture representing five classes of phytoplankton. Statistical analyses revealed that both the growth rate and the factor limiting growth (NO₃^-, NH₄⁺, PO₄^3- or light) could alter C:ATP, RNA: ATP, C:chl a and RNA:chl a ratios by amounts which were large compared to measurement error. An analysis of variance of the batch culture results indicated that both species and the source of inorganic nitrogen (NO₃^-, or NH₄⁺) had a significant effect on C:ATP and RNA:ATP ratios. Light had less of an influence on C:ATP and RNA:ATP ratios than on C:chl a and RNA:chl a ratios, and for this reason we feel that phytoplankton C and RNA concentrations can be estimated with greater reliability from ATP than from chl a measurements. The range of C:ATP and RNA:ATP values found for T. weissflogii under a variety of growth conditions was similar to that for the six additional species grown in batch culture, suggesting that this range of values is indicative of the extremes likely to occur in living cells. Our results and additional data in the literature indicate that phytoplankton C and RNA concentrations can be estimated to within a factor of two by multiplying ATP concentrations by 311 and 35, respectively, in N limited systems, and by 341 and 36, respectively in PO₄^3- limited systems.     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.     

Reverse electron flow in chloroplasts

Energy dependent reverse electron flow reactions in isolated thylakoids provide a unique tool to study, in the dark, the coupling between the ATP synthase, proton transport and the electron transfer system. Appropriate experimental conditions have been established to follow experimentally the following reactions:

1. ATP driven proton uptake into the inner-thylakoid space, which requires preactivation of the ATP synthase.
2. ATP driven reverse electron transport, which involves proton transport as an intermediate, and results in the reduction of Q_A by an externally added electron donor.
3. ATP driven luminescence, which requires the presence of an oxidized partner on the water side of photosystem II, and involves electron transport from Q_B to Q_A.
4. ΔpH driven reverse electron flow, which does not require the participation of the ATP synthase, and uses reduced intermediates between the two photosystems as electron donors for the reduction of Q_A.
5. ΔpH driven luminescence which again uses reduced intermdiates between the two photosystems as electron donors for Q_A reduction, and requires the presence of an oxidized partner on the water side of photosystem II.

Several of these reactions have been shown to occur in intact chloroplasts and may provide an important regulatory mechanism in vivo.     

Stoichiometry of Energy Coupling by Proton-Translocating ATPases: A History of Variability

One of the central energy-coupling reactions in living systems is the intraconversion of ATP with a transmembrane proton gradient, carried out by proton-translocating F- and V-type ATPases/synthases. These reversible enzymes can hydrolyze ATP and pump protons, or can use the energy of a transmembrane proton gradient to synthesize ATP from ADP and inorganic phosphate. The stoichiometry of these processes (H⁺/ATP, or coupling ratio) has been studied in many systems for many years, with no universally agreed upon solution. Recent discoveries concerning the structure of the ATPases, their assembly and the stoichiometry of their numerous subunits, particularly the proton-carrying proteolipid (subunit c) of the F_O and V₀ sectors, have shed new light on this question and raise the possibility of variable coupling ratios modulated by variable proteolipid stoichiometries.     

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.     

ATP-induced lysis of rat parotid secretory granules: Possible role of ATP in exocytotic release

Secretory vesicles isolated from a variety of mammalian tissues are known to lyse and thereby release their secretory products when exposed to ATP. This process, which will be termed ATP-induced lysis, has been studied most extensively using adrenal chromaffin-granule preparations. We report here that ATP causes the lysis of a highly purified preparation of rat parotid secretory granules. The rate of granule lysis was measured spectrophotometrically, and ATP-induced lysis was expressed as the increase in the rate of lysis (r = % lysis per min) when ATP was added. This lytic process was characterized with respect to pH, temperature, osmolarity, and the ionic composition of the media ATP-induced lysis of parotid granules was found to have the following properties in common with the extensively characterized chromaffin-granule process:

• 1 It is a saturable function of ATP with half-maximal rates observed at 0.5 ± 0.1 mM ATP.
• 2 It is temperature dependent, eg, r = 6.1 ± 2.1%/min at 30°C vs 12.2 ± 2.5%/min at 37°C.
• 3 It is inhibited in hyperosmotic media, eg, r = 5.3 ± 0.3%/min at 0.3 OsM vs 0.8 ± 0.2%/min at 0.4 OsM.
• 4 It shows a nucleotide preference of ATP = GTP > ADP > AMP > CTP = ITP.
• 5 It has an anion requirement.

The above findings, combined with reports of ATP-induced lysis of cholinergeric, insulin, and posterior-pituitary vesicles, imply that ATP-induced lysis may reflect an ATP-dependent property of all secretory vesicles, and as such, this vesicle property could play a similar role in each exocytotic release process. Using a model system, Miller and Racker [22] made a surprising finding that the extent to which liposomes fuse with a black lipid membrane depends on the osmotic gradient across the vesicle membrane. In view of the osmotic dependence of ATP-induced lysis in this and other secretory-vesicle preparations, we postulate that ATP may prime secretory vesicles for fusion with the plasma membrane by inducing and/or maintaining an osmotic gradient across the vesicle membrane.     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: Kinetic characterization of NEDD8 activating enzyme

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous “E1s” initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [³²P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [³²P]PPi and then measuring [³²P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [³²P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.     

Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose-1-phosphate uridyl transferase and uridine diphospho-galactose-4-epimerase.

• 1 Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.
• 2 Added extracellular Gal-1-P, G-1-P, UDPGal and UPDG do not affect enzyme activities in whole cells.
• 3 The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.
• 4 Galactokinase activity is inhibited by G-6-P and Gal-1-P, but not by glucose, G-1-P, UDPG, UDPGal, UTP or NAD⁺. It is inhibited by ATP and ADP in high concentration.
• 5 Galactose-1-phosphate uridyl transferase activity is inhibited by G-1-P, G-6-P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD⁺, or galactose.
• 6 Uridine diphospho-galactose-4-epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD⁺ and possibly by Gal-1-P. It is unaffected by G-1-P, G-6-P.
• 7 The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.
• 8 Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.
• 9 Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal-u-¹⁴C.
• 10 ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

     

An evaluation of the performance of ten commercial luminometers

An assessment has been carried out of the relative performance of ten instruments for quantification of adenosine triphosphate (ATP) by the firefly luciferase assay. The instruments evaluated were Amersham Amerlite Analyser, Dynatech Tube Luminometer, Dynatech Multiplate Luminometer, Dynatech Camera Luminometer, Hamilton Lumicon, LKB 1250 Luminometer, LKB 1251 Luminometer, Lumac Biocounter M2010A, Turner 20 TD Luminometer and a prototype version of the CLEAR Speed Tech 2000. An 800-fold difference in sensitivity was found between the most sensitive (Lumac, Turner) and the least sensitive (Dynatech Tube) of the conventional instruments. The Dynatech Camera Luminometer which worked on a completely different principle to the other instruments was about 5000 times less sensitive than the best of the photomultiplier tube instruments. The relative sensitivity of the instruments was maintained regardless of whether solutions of ATP in water or trichloroacetic acid extracts of bacteria were analysed. An analysis of 960 ATP bioluminescence assays showed that data obtained from such measurements are normally distributed.     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

Quantitative analysis of depolarization-induced ATP release from mouse brain synaptosomes: External calcium dependent and independent processes

Summary We and others have shown previously that ATP is secreted from mouse brain synaptosomes following depolarization of the membrane by high [K⁺] ₀ and the time course can be monitored accurately by measuring the light emitted from luciferin-luciferase included in the reaction medium. In the present work we have evaluated the relative importance of [Ca²⁺] ₀ and membrane potential on the ATP secretion process by modelling the time course of ATP release under different conditions. After correction of the records for destruction of released ATP by synaptosomal ecto-ATPase activity, we found that ATP secretion occurs by an apparent first order process. We also established that, in addition to the classical [Ca²⁺] ₀ -dependent mode, ATP secretion also occurred in the absence of extracellular calcium ([Ca²⁺] ₀ < 1 m). Upon lowering the extracellular Ca²⁺ concentration, both the rate and the extent of ATP secretion decreased. To assess the contribution of membrane potential to the release rate we measured ATP secretion at membrane potentials determined by extracellular [K⁺] ₀ (or [Rb⁺] ₀ ) as defined by the distribution of the carbocyanine dye, diSC₃(5). Rate constants computed from measured secretion curves revealed that this parameter was essentially independent of membrane potential in the absence of [Ca²⁺] ₀ . Noise analysis of the light signal showed that the variance increased upon stimulation by high [K⁺] ₀ , suggesting that both modes of secretion are quantal. Thus, we conclude that the rate of ATP secretion from nerve terminals depends upon Ca²⁺ entry but not on membrane potential, per se