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PubMed1986

Control of membrane permeability by external ATP in mammalian cells: isolation of an ATP-resistant variant from Chinese hamster ovary cells.

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.

Adenosine Triphosphate↗

PubMed1987

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve.

The ATP hydrolysis dependent Na+-Na+ exchange of reconstituted shark (Na+ + K+)-ATPase is electrogenic with a transport stoichiometry as for the Na+-K+ exchange, suggesting that translocation of extracellular Na+ is taking place via the same route as extracellular K+. The preparation thus offers an opportunity to compare the sided action of Na+ and K+ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K+. With Na+ but no K+ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K+ in concentrations of 50 microM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K+ also gives biphasic ATP-activation curves, however, only when the K+ concentration is 50 mM or higher (Na+ + K+ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E2(Na2+) and E2(K2+), respectively, and in which the dephosphorylation of E2-P is rate limiting in the presence of Na+ (but no K+) extracellular, whereas in the presence of extracellular K+ it is the deocclusion of E2(K2+) which is rate limiting.

Adenosine Triphosphate↗

PubMed1994

ATP-dependent transport of the linear renin-inhibiting peptide EMD 51921 by canalicular plasma membrane vesicles of rat liver: evidence of drug-stimulatable ATP-hydrolysis.

Certain peptide drugs, such as the linear hydrophobic renin-inhibitor EMD 51921, are rapidly eliminated via the bile. At the sinosoidal membrane of liver cells EMD 51921 is taken up via a sodium-independent carrier-mediated mechanism, competing for the uptake of bile acids. Until now, the mechanisms of biliary excretion of EMD 51921 were unknown. In this study we describe an ATP-dependent transport system for the enzymatically and metabolically stable hydrophobic linear renin-inhibiting peptide EMD 51921. The ATP-dependent uptake into the osmotic reactive intravesicular space is saturable (Km 12 microM, Vmax 663 pmol/min per mg protein), temperature dependent and specifically requires ATP. Transport is inhibited by vanadate but not by ouabain, EGTA or NaN3, and does not function in basolateral plasma membrane vesicles. Transport is not altered in canalicular membrane vesicles isolated from Tr- rats lacking the canalicular ATP-dependent transport of cysteinyl leukotrienes and related anions. Transport is inhibited by taurocholate, a typical substrate of the canalicular ATP-dependent bile acid transporter, but also by vincristine and daunomycin, substrates of P-glycoproteins. EMD 51921, however, only inhibits the uptake of taurocholate, whereas the transport of daunomycin is not influenced. Taurocholate and EMD 51921 are mutually non- or un-competitive transport inhibitors. Incubation of rat liver canalicular membranes with micromolar concentrations of EMD 51921 resulted in a 1.8-2.5-fold increase in the rate of ATP-hydrolysis. In contrast, ATP-hydrolysis was not affected by fragments of the peptide that are not transported in an ATP-dependent manner. The apparent Km value (EMD) for ATP-hydrolysis is 68 microM. Vmax is 0.032 U/mg protein. ATPase activity is pH dependent. Stimulation of ATP-hydrolysis is inhibited by vanadate, NEM, hydroxymercuribenzoate and ascorbate, but is not affected by ouabain, EGTA or NaN3. EMD 51921 does not stimulate the ATPase activity of the Na+/K(+)-ATPase isolated from kidney medulla. The EMD-stimulatable ATPase seems to be distinct from the glutathione-S-conjugate stimulatable ATPase and the mdr 1a/b gene products and differs in its characteristics from that of the canalicular ecto-ATPase.

Adenosine Triphosphatases↗

PubMed1982

Effect of arylazido aminopropionyl ATP (ANAPP3), on ATP responses of isolated guinea pig smooth muscle.

Arylazido aminopropionyl ATP (ANAPP3), a photoaffinity analogue of adenosine 5'-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP3 (30 microM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 microM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 microM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP3 (30 and 100 microM) failed to antagonize relaxant responses to ATP (1.0 - 1000 microM) in fundic strips. In addition ANAPP3 failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP3 does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.

Adenosine Triphosphate↗

PubMed2005

Interactions of caged-ATP and photoreleased ATP with alkaline phosphatase.

Photolytic release of ATP from inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP (NPE-caged ATP) provides a means to reveal molecular interactions between nucleotide and enzyme by using infrared spectroscopy. Reaction-induced infrared difference spectra of bovine intestinal alkaline phosphatase (BIAP) and of NPE-caged ATP revealed small structural alterations on the peptide backbone affecting one or two amino-acid residues. After photorelease of ATP, the substrate could be hydrolyzed sequentially by the enzyme producing three Pi, adenosine, and the photoproduct nitrosoacetophenone. It was concluded that NPE-caged ATP could bind to BIAP prior to the photolytic cleavage of ATP and that Pi could interact with BIAP after photolysis of NPE-caged ATP and hydrolysis, yielding infrared spectra with distinct structure changes of BIAP. This suggests that the molecular mechanism of ATP hydrolysis by BIAP involved small structural adjustments of the peptide backbone in the vicinity of the active site during ATP hydrolysis which continued during Pi binding.

Adenosine Triphosphate↗

PubMed2006

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding.

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.

Adenosine Triphosphate↗

PubMed2002

Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP-sensitive K+ channels.

Inhibition of ATP-sensitive K(+) (K(ATP)) channels by ATP, a process presumably initiated by binding of ATP to the pore-forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer-specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch-clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP-binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of K(ATP) channels.

Adenosine Triphosphate↗

PubMed1998

ATP-sensitive K+ channels in cardiac muscle from cold-acclimated goldfish: characterization and altered response to ATP.

ATP-sensitive potassium channels (K(ATP)) play an important, if incompletely defined, role in myocardial function in mammals. With the discovery that K(ATP) channels are also present at high densities in the hearts of vertebrate ectotherms, speculation arises as to their function during periods of cold-acclimation and depressed ATP synthesis. We used single-channel and intracellular recording techniques to examine the possibility that channel activity would be altered in cardiac muscle from goldfish (Carassius auratus) acclimated at 7+/-1 degrees C relative to control (21+/-1 degrees C). As previously observed in mammals, K(ATP) channels in isolated ventricular myocytes were inwardly rectified with slope conductances of 63 pS. However, channel mean open-time and overall open-state probability (Po) were significantly increased in cells from the cold-acclimated animals. In addition, K(ATP) channels in cells from fish acclimated at 7 degrees were nearly insensitive to the inhibitory effects of 2 mM ATP, whether studied at 7 or at 21 degrees C. Transmembrane action potential duration (APD) in hearts of cold-acclimated fish studied at 21 degrees was significantly shorter than that observed in hearts of warm-acclimated fish at the same temperature; this difference was eliminated by the K(ATP) channel antagonist glibenclamide (5 microM). These data suggest that K(ATP) channels in the hearts of cold-acclimated animals are more active and less sensitive to ATP-inhibition than those in warm-acclimated fish, possibly reflecting a functional adaptation to promote tolerance of low temperatures in this species.

Acclimatization↗

PubMed1984

Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1.

The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1988

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase.

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗

PubMed1990

Inhibition of recA protein promoted ATP hydrolysis. 2. Longitudinal assembly and disassembly of recA protein filaments mediated by ATP and ADP.

There are at least two major conformations of recA nucleoprotein filaments formed on poly-(deoxythymidylic acid) [poly(dT)], one stabilized by ATP [or adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S)] and one stabilized by ADP. Assembly of filaments in the ATP conformation is much faster than assembly in the ADP conformation. A third conformation may be present in the absence of nucleotides. The ATP and ADP conformations are mutually exclusive. When a mixture of ATP and ADP is present, recA protein binding is a function of the ADP/ATP ratio. Complete dissociation is observed when the ratio becomes 1.0-1.5. When a mixture of ATP and ADP is present at the beginning of a reaction, a transient phase lasting several minutes is observed in which the system approaches the state characteristic of the new ADP/ATP ratio. This phase is manifested by a lag in ATP hydrolysis when ATP is added to preformed ADP filaments, and by a burst in ATP hydrolysis in all other cases. More than 15 ATPs are hydrolyzed per bound recA monomer during the burst phase. The transient phase reflects an end-dependent disassembly process propagated longitudinally through the filament, rather than a slow conformation change in individual recA monomers or a slow exchange of one nucleotide for the other. The hysteresis exhibited by the system provides a number of insights relevant to the mechanism of recA-mediated DNA strand exchange.

Adenosine Diphosphate↗

PubMed1978

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.

Actins↗

PubMed2006

Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis.

The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.

Adenosine Triphosphate↗

PubMed1998

A nucleotide-binding domain of porcine liver annexin VI. Proteolysis of annexin VI labelled with 8-azido-ATP, purification by affinity chromatography on ATP-agarose, and fluorescence studies.

Porcine liver annexin VI (AnxVI) of Mr 68.000 is an ATP-binding protein as evidenced by specific and saturable UV-dependent labelling with 8-azido-[gamma-32P]ATP or the fluorescent analog of ATP, 2'-(or 3')-O-(2,4,6-trinitrophenyl)adenosine triphosphate and by binding of AnxVI to ATP-agarose. These characteristics of purified AnxVI were used to identify and characterize preliminary nucleotide-binding domain of the protein. AnxVI labelled with 8-azido-ATP was subjected to limited proteolysis and the proteolytic fragments of AnxVI that retained the covalently-bound nucleotide were separated by means of gel electrophoresis and visualized by exposure of the gel to a phosphor storage screen. It was found that the AnxVI proteolytic fragments of Mr 34-36.000 and smaller retained the nucleotide. In a reciprocal experiment, AnxVI was digested with proteolytic enzymes and in an ATP eluate from an ATP-agarose column protein fragments of similar Mr to these labelled with 8-azido-ATP were identified. The extent of AnxVI labelling with 8-azido-ATP and the distribution of proteolytic fragments varied upon calcium concentration. These results lead to the conclusion that there is a nucleotide-binding domain within the AnxVI molecule that is functionally similar to the nucleotide-binding domains of other nucleotide-binding proteins. The nucleotide-binding domain is located close to the tryptophan residue 343 of AnxVI and in close vicinity to the Ca2+- and phospholipid-binding sites of the protein. This is confirmed by the observation that the tryptophan fluorescence intensity of AnxVI decreases in the presence of a fluorescence analog of ATP in a calcium-dependent manner, due to the quenching properties of the nucleotide and/or fluorescence energy transfer from AnxVI tryptophan to fluorophore. Both processes were modulated by the presence of phospholipid molecules.

Adenosine Triphosphate↗

PubMed2005

ATP-driven stepwise rotation of FoF1-ATP synthase.

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.

Adenosine Triphosphate↗

PubMed2001

Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin.

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Adenosine Triphosphate↗

PubMed1996

Identification of the ATP binding domain of recombinant human 40-kDa 2',5'-oligoadenylate synthetase by photoaffinity labeling with 8-azido-[alpha-32P]ATP.

Three isoforms of the interferon-inducible 2',5'-oligoadenylate (2-5A) synthetase that require double-stranded RNA have been isolated and cloned. However, identification of the amino acid(s) of 2-5A synthetase directly interacting with ATP is crucial to the elucidation of the mechanism of the enzymatic conversion of ATP to 2',5'-oligoadenylates by 2-5A synthetase. Recombinant human 40-kDa 2-5A synthetase has been expressed as a glutathione S-transferase fusion protein in E. coli and purified to near homogeneity in milligram quantities. The azido photoprobe, 8-azido-[alpha-32P]ATP, has been used to identify the ATP binding domain of the recombinant human 40-kDa 2-5A synthetase. Specific covalent photoincorporation of 8-azido-[alpha-32P]ATP into the 2-5A synthetase, tryptic digestion of the covalently 32P-labeled enzyme, isolation of the photolabeled phosphopeptide by metal (Al3+) chelate chromatography, and high pressure liquid chromatography identified a 32P-pentapeptide, which has been assigned to the ATP binding domain of 2-5A synthetase. The radioactive pentapeptide has the sequence D196FLKQ200 in which the photoprobe, 8-azido-[alpha-32P]ATP, chemically modified the amino acid lysine 199. The catalytic importance of Lys199 was further established by mutation of lysine 199 to arginine 199 and histidine 199 using site-directed mutagenesis. The K199R and K199H recombinant human 40-kDa 2-5A synthetase mutants bind 8-azido-ATP and the allosteric activator, poly(I) poly(C) but are enzymatically inactive. These photoaffinity labeling and mutation data strongly suggest that lysine 199 is essential for the formation of a productive 2-5A synthetase-ATP-double-stranded RNA complex for the enzymatic conversion of ATP to 2-5A.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E2ATP site leads to inhibition of Na+/K+-ATPase. Analysis of structural requirements of fluorescent ATP derivatives with a Koshland-Némethy-Filmer model of two interacting ATP sites.

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (Kd = 0.7 microM) and to the E2ATP site (Kd = 210 microM), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (Kd = 200 microM) than to the E2ATP binding site (Kd = 80 microM). 3'(2')-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (Kd = 10 microM). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2'(3')-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site.

Adenosine↗