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PubMed1987

The acceleration of Na+,K+-ATPase activity by ATP and ATP analogues.

Since Na+,K+-ATPase (EC 3.6.1.3) of pig kidney modified with a fluorescent sulfhydryl reagent, N-[p-(2-benzimidazolyl) phenyl]maleimide, at Cys-964 of the alpha-chain showed ATP-dependent, reversible, and dynamic fluorescence changes (Nagai, M., Taniguchi, K., Kangawa, K., Matsuo, S., Nakamura, S., and Iida, S. (1986) J. Biol. Chem. 261, 13197-13202), we studied the conformational change during Na+,K+-ATPase reaction using the modified enzyme. The addition of K+ to the enzyme increased the fluorescence intensity to 2% in the presence of 160 mM Na+ and 3 mM Mg2+ (K0.5 = 16.4 mM). Addition of low concentrations of ATP immediately increased the intensity to 3.2% (K0.5 less than 0.1 microM) to accumulate fully K+-bound enzyme in the presence of 43 mM K+ with Na+ and Mg2+, but further addition of higher concentrations of ATP diminished the increase (K0.5 = 120 microM). After exhaustion of ATP, the fluorescence intensity decreased to -0.4% (K0.5 = 0.3 microM) and -2% (K0.5 = 20 microM), respectively, in the presence of low and high concentrations of ADP produced from ATP. High concentrations of ATP accelerated Na+,K+-ATPase activity with a simultaneous increase in the amount of ADP-sensitive phosphoenzyme irrespective of the modification. Adenylyl imidodiphosphate and ADP accelerated Na+,K+-ATPase activity in the presence of 2.7 microM ATP by decreasing the extent of the fluorescence without affecting the amount of phosphoenzyme, irrespective of the modification. These data suggest that Na+,K+-ATPase activity was accelerated due to the acceleration of the breakdown of K+-bound enzyme by high concentrations of ATP and ATP analogues.

Adenosine Diphosphate↗

PubMed1987

Characterization of 2'(3')-trinitrophenyl-ATP as an inhibitor of ATP-dependent initiation complex formation between the DNA polymerase III holoenzyme and primed DNA.

We have identified 2'(3')-trinitrophenyl-ATP to be an inhibitor of the ATP-dependent initiation complex formation reaction between the Escherichia coli DNA polymerase III holoenzyme and primed DNA. The inhibitor is specific for the initiation stage; once initiation complexes are formed the subsequent elongation reaction is unaffected. Three ATP-dependent DNA polymerase III holoenzyme reactions can be independently assayed: the ATP-dependent formation of initiation complexes, ATP binding, and the primed DNA-dependent hydrolysis of ATP. Trinitrophenyl ATP inhibits all three reactions to a similar extent with an apparent Ki between 6 and 15 microM in the presence of 5 microM ATP. This suggests all of these reactions are related and that they proceed through a common ATP-binding site. We include an improved purification of the DNA polymerase III holoenzyme in this report.

Adenosine Triphosphate↗

PubMed1984

Localization of the high-affinity binding site for ATP on the membrane-bound chloroplast ATP synthase.

The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.

Adenosine Diphosphate↗

PubMed1976

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.

Adenosine Triphosphate↗

PubMed1995

ATP depletion and inactivation of an ATP-sensitive taurine channel by classic ion channel blockers.

Cell volume regulation in different cell types is mediated in part by plasma membrane channel(s) that allow taurine and other important intracellular organic osmolytes to efflux from the cell. Previous studies have demonstrated that intracellular ATP is required for activation of a volume-sensitive taurine-permeable channel. The present study examined the relation between cellular ATP and ADP concentrations and swelling-induced [14C]taurine efflux and anion current (whole-cell patch-clamp) after exposure of isolated skate (Raja erinacea) hepatocytes to metabolic poisons and a series of ion channel blockers. When intracellular ATP content was lowered with gradually increasing concentrations of 2,4-dinitrophenol, a sigmoidal relation between ATP content and volume-activated [14C]taurine efflux was observed. Taurine efflux was progressively inhibited over a relatively narrow range of intracellular ATP levels, indicating that physiologic alterations in cellular nucleotides may modulate the opening of the channel. Surprisingly, the inhibition of [14C]taurine efflux by a number of ion channel blockers [glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoate, diphenylamine-2-carboxylate, ketoconazole, gossypol, niflumic acid, and quinine] was related to a decrease in cellular ATP concentrations and ATP/ADP ratios, rather than to a direct interaction with the channel. In contrast, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and pyridoxal-5-phosphate inhibited volume-activated anion channels but had no effect on cellular ATP levels. These findings suggest multiple sites for regulation of volume-sensitive osmolyte channels and indicate that some putative ion channel blockers may actually alter the activity of ATP-regulated transporters by depleting cellular ATP.

2,4-Dinitrophenol↗

PubMed1994

Solving the common problem: matching ATP synthesis to ATP demand during exercise.

Biologists have long known that rates of adenosine triphosphate utilization and production need to be extremely closely balanced in all cells at all work rates. To put it in molecular rather than molar terms, in human muscle engaged in a 15-min work protocol, some 3.3 x 10(20) ATP molecules g-1 are used and resynthesized at close to 100 times the resting cycling rates before fatigue occurs. However, during this interval only a 20-25% decrease in the ATP pool is sustained. Review of recent studies as to how such remarkable regulatory precision is achieved suggests that in resting muscle, myosin behaves as a latent catalyst for which the full catalytic capacity is (a) realized with the arrival of its activator signal (Ca2+) and (b) attenuated with reaction products. These proactive controls, initiated at the onset of ATP utilization, set the required flux through ATP-producing pathways. For any given enzyme step in ATP-producing pathways, reaction velocity (upsilon) becomes the independent parameter, whereas substrate concentration ([s], the dependent parameter) must be adjusted accordingly. Because the dynamic range for muscles (change from resting to maximum ATP turnover rates) can exceed 100-fold, in many studies of working muscle the percentage change in ATP turnover rate usually greatly exceeds the percentage change in substrate concentrations. These kinds of observations are not easily explained by current metabolic regulation models, but are consistent with pathway enzymes behaving as latent catalysts in resting muscle. In this view, the unmasking of such latent catalytic potential is the main explanation for how large changes in upsilon can be achieved with modest (sometimes immeasurable) changes in [s]. In terms of the basic enzyme velocity equation, Vmax = kcat.e0 for pathways of ATP utilization and of ATP synthesis, the dominant (coarse level) regulation is achieved through e0 adjustments, whereas fine-tuning of flux is achieved by effective kcat adjustment.

Adenosine Diphosphate↗

PubMed2026

Beyond NAD Depletion: SARM1-Induced ATP Collapse Involves Direct ATP Degradation and Mitochondrial Dysfunction and Is Pharmacologically Reversible.

Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is an inducible NAD-consuming enzyme and execution factor in axon degeneration. Rapid ATP collapse after SARM1 activation, however, is not fully explained by NAD depletion alone. We used SARM1-overexpressing HEK293 cells and the cell-permeant activator CZ-48 to examine SARM1-induced non-apoptotic cell death, termed sarmoptosis. CZ-48 induced cell death that was suppressed by HSP90/70-annotated ATP-competitive compounds, especially geldanamycin and VER-155008 (VER), without reducing SARM1 abundance. VER preserved NAD and ATP during SARM1 activation but failed to rescue FK866-mediated NAD starvation, thereby distinguishing CZ-48/SARM1-driven cytotoxicity from generic NAD depletion. In cell-free assays, purified SARM1 reduced ATP levels; this effect was enhanced by SARM1's activator NMN and attenuated by its pharmacological inhibitors, although the in vitro activity was modest and the reaction products remain to be identified. ATPase-related perturbations, including thapsigargin and bafilomycin A1, also protected cells from CZ-48-induced death, further supporting a central role for ATP collapse in sarmoptosis. iTRAQ proteomics, MitoSOX Red staining, and DiOC6(3) staining revealed that CZ-48 treatment was associated with mitochondrial and metabolic remodeling, mitochondrial ROS accumulation, and mitochondrial depolarization, all of which were mitigated by VER. Collectively, these findings support a convergent ATP-collapse model in which SARM1 activation promotes NAD depletion, directly consumes ATP, and is associated with mitochondrial dysfunction that may amplify ATP-production failure.

ATP collapse↗

PubMed2003

The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p.

The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a Km of 0.6 mm and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1.7), indicating that the functional state is a dimer. Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD. The nucleotide composition of trapped intermediate states was determined using [alpha-32P]ATP and [gamma-32P]ATP. Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode. Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides.

ATP-Binding Cassette Transporters↗

PubMed2004

Reversible assembly of the ATP-binding cassette transporter Mdl1 with the F1F0-ATP synthase in mitochondria.

The half-ABC transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins. The physiological role of the peptides released from mitochondria is currently not understood. Here, we have analyzed the oligomeric state of Mdl1 in the inner membrane and demonstrate nucleotide-dependent binding to the F(1)F(0)-ATP synthase. Mdl1 forms homo-oligomeric, presumably dimeric complexes in the presence of ATP, but was found in association with the F(1)F(0)-ATP synthase at low ATP levels. Mdl1 binds membrane-embedded parts of the ATP synthase complex after the assembly of the F(1) and F(0) moieties. Although independent of Mdl1 activity, complex formation is impaired upon inhibition of the F(1)F(0)-ATP synthase with oligomycin or N,N'-dicyclohexylcarbodiimide. These results are consistent with an activation of Mdl1 upon dissociation from the ATP synthase and suggest a link of peptide export from mitochondria to the activity of the F(1)F(0)-ATP synthase and the cellular energy metabolism.

ATP-Binding Cassette Transporters↗

PubMed2001

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

ATP-Binding Cassette Transporters↗

PubMed1984

Cloning of cDNA sequences for murine ATP-citrate lyase. Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver.

Mouse liver mRNA that was enriched in sequences coding for ATP-citrate lyase by polysome immunoadsorption was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli RR1. Twenty-seven plasmids containing putative cDNA sequences for ATP-citrate lyase were identified by differential hybridization with single-stranded 32P-cDNAs synthesized from immunopurified mRNA, sucrose gradient-purified ATP-citrate lyase mRNA, and mRNA isolated from the livers of mice that were nutritionally induced or de-induced for ATP-citrate lyase biosynthesis. A subgroup of five recombinant plasmids was characterized further in hybridization-selection experiments. Each of these plasmids positively selected ATP-citrate lyase mRNA as determined by in vitro translation and specific immunoprecipitation. The length of ATP-citrate lyase mRNA was estimated to be 4900 bases in a Northern blot analysis. A 32P-cDNA probe derived from a 1500-base pair insert was used to investigate the basis for the 20-30-fold induction of ATP-citrate lyase that occurs when starved animals are fed a high carbohydrate/low fat diet. Dot-blot hybridization analysis disclosed that the relative content of liver ATP-citrate lyase mRNA increased 25-fold after 15 h of refeeding, indicating that the synthesis of the lipogenic enzyme is controlled at a pretranslational level in the nutritional paradigm.

ATP Citrate (pro-S)-Lyase↗

PubMed1993

Interaction of Ant-ATP with tubulin: evidence for ATP competition for the GTP E-site on tubulin.

The interaction of the ribose-modified ATP analogue 3'-O-anthraniloyl adenosine 5'-triphosphate (Ant-ATP) with tubulin has been studied using steady-state fluorescence techniques. This analogue inhibits the polymerization of tubulin induced by ATP or GTP. When this analogue binds to tubulin, an increase in the fluorescence intensity of the analogue and a blue shift of about 10 nm in the emission maximum have been observed. It has been found that Ant-ATP binds to tubulin at a single binding site in the hydrophobic region with a dissociation constant of 0.5-1.0 microM. It has been possible to restore the fluorescence emission intensity of the tubulin-bound analogue to that of the free ligand with a concomitant shift in the wavelength of emission maximum to that of the free analogue by displacing the analogue with ATP or GTP. These results can be interpreted to suggest that ATP and GTP compete for the Ant-ATP binding site and that ATP binds at the GTP exchangeable site (E-site).

Adenosine Triphosphate↗

PubMed2002

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran.

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.

Adenosine Triphosphate↗

PubMed1995

Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Adenosine Triphosphatases↗

PubMed2002

The molecular mechanism of ATP synthesis by F1F0-ATP synthase: a scrutiny of the major possibilities.

A critical goal of metabolism in living cells is the synthesis of adenosine triphosphate (ATP). ATP is synthesized by the enzyme F1F0-ATP synthase. This enzyme, the smallest-known molecular machine, couples proton translocation through its membrane-embedded, hydrophobic domain, F0, to the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) in its soluble, hydrophilic headpiece, F1. Animals, plants and microorganisms all capture and utilize energy by this important chemical reaction. How does it occur? The binding change mechanism and the torsional mechanism of energy transduction and ATP synthesis are two mechanisms that have been proposed in the literature. According to the binding change mechanism (which considers reversible catalysis and site-site cooperativity), energy is required primarily for release of synthesized ATP, but not for its synthesis. On the other hand, according to the torsional mechanism (which considers an irreversible mode of catalysis and absence of cooperativity), all the elementary steps require energy, and the ion-protein interaction energy obtained from the ion gradients is used to synthesize ATP, for Pi binding, and for straining the beta-epsilon bond in order to enable ADP to bind. The energy to release preformed ATP from the tight catalytic site (betaDP) is provided by the formation of the beta-epsilon ester linkage. First, the central features of these mechanisms are clearly delineated. Then, a critical scrutiny of these mechanisms is undertaken. The predictions of the torsional mechanism are listed. In particular, how the torsional mechanism deals with the specific difficulties associated with other mechanisms, and how it seeks to explain a wealth of structural, spectroscopic, and biochemical data is discussed in detail. Recent experimental data in support of the mechanism are presented. Finally, in view of the molecular machine nature of energy transduction, the indispensability of applying engineering tools at the molecular level is highlighted. This paves the way for the development of a new field: Molecular Physiological Engineering.

Adenosine Triphosphate↗

PubMed1993

ATP formation and ATP hydrolysis during fatiguing, intermittent stimulation of different types of single muscle fibres from Xenopus laevis.

This report describes changes of the rate of ATP hydrolysis in single, intact muscle fibres during the development of fatigue induced by intermittent tetanic stimulation. High (type 3) and low (type 1) oxidative muscle fibres dissected from the iliofibularis muscle of Xenopus laevis were studied at 20 degrees C. The rate of ATP hydrolysis was calculated during different time intervals from changes in the content of nucleotides, creatine compounds and lactate, as well as lactate efflux and oxygen uptake. During the first phase of intermittent stimulation, phosphocreatine is fully reduced while the rate of oxygen consumption increases to its maximum, the lactate content increases to a maximum level, and a small amount of IMP is formed; the rate of ATP hydrolysis in type 3 fibres is constant while force decreases, whereas the rate decreases approximately in proportion to force in type 1 fibres. After the first phase, the rate of ATP hydrolysis in type 3 fibres decreases slightly and the fibres reach a steady metabolic state in which the rates of ATP formation and hydrolysis are equal; in type 1 fibres a drastic change of the rate of ATP hydrolysis occurs and a steady metabolic state is not reached. On the basis of the time courses of the metabolic changes, it is concluded that the rate of ATP hydrolysis in type 3 fibres is reduced by acidification and/or a reduced calcium efflux from the sarcoplasmic reticulum, whereas in type 1 fibres inorganic phosphate and/or acidification inhibit the rate initially and ADP is a likely candidate to explain the drastic fall of the rate of ATP hydrolysis during late phases of fatiguing stimulation.

Adenosine Diphosphate↗

PubMed1995

Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: characterization using [32P]3'-O-(4-benzoyl)benzoyl ATP, a photoaffinity label.

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.

Adenosine Triphosphate↗

PubMed1989

ATP-sensitive potassium channels in adult mouse skeletal muscle: characterization of the ATP-binding site.

Single K+-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160 mM KCl solutions were blocked reversibly by 10 microM internal ATP and thus identified as ATP-sensitive K+ channels. The channels were also blocked reversibly by mM concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K+ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5 mM chloramine-T, 50 mM hydrogen peroxide or 2 mM N-ethylmaleimide (NEM). Large-conductance Ca-activated K+ channels were not affected by these reagents. The presence of 1 mM internal ATP prevents the irreversible inhibition of ATP-sensitive K+ channels by NEM. The results suggest that internal Na+ ions increase the affinity of the ATP-sensitive K+ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.

Adenosine↗