Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.     

Source of rapidly labeled ATP tightly to non-catalytic sites on the chloroplast ATP synthetase

Bound [32P]ATP is found on deenergized, washed chloroplast thylakoids which were illuminated in the presence of ADP and [32P]Pi. Tight binding of [32P]ATP occurred both during and after energization. Different classes of bound [32P]ATP were distinguished on the basis of their rates of formation, susceptibility to hexokinase and displacement by unlabeled ATP. 1. The rates of formation and discharge of the rapidly labeled tightly bound ATP class were much lower than that of ATP formation. The level of this bound ATP saturates at lower concentrations of substrates than does the rate of phosphorylation. Unlabeled ATP, present in the reaction medium, displaces the rapidly labeled tightly bound ATP without affecting the rate of phosphorylation. 2. We therefore conclude that the rapidly labeled bound ATP class does not fulfill the requirements expected for a catalytic intermediate and that the nucleotide tight binding site(s) on the ATP synthetase differ from the catalytic site(s) for ATP formation. 3. Since the rapidly labeled tightly bound [32P]ATP is not abolished by high concentrations of hexokinase, but is nevertheless displaced by exogenous ATP, we propose that tight binding of ATP to non-catalytic sites occurs via a free species of newly synthesized ATP which diffuses slowly to the medium from a space accessible to ATP but not to hexokinase.     

ATP as a presynaptic modulator

There is considerable evidence that ATP acts as a fast transmitter or co-transmitter in autonomic and sensory nerves mostly through activation of ionotropic P2X receptors but also through metabotropic P2Y receptors. By analogy, the observations that ATP is released from stimulated central nervous system (CNS) nerve terminals and that responses to exogenously added ATP can be recorded in central neurons, lead to the proposal that ATP might also be a fast transmitter in the CNS. However, in spite of the robust expression of P2 receptor mRNA and binding to P2 receptors in the CNS, the demonstration of central purinergic transmission has mostly remained elusive. We now review evidence to suggest that ATP may also act presynaptically rather than solely postsynaptically in the nervous system.     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

Insulin-degrading enzyme hydrolyzes ATP

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.     

ATP synthase: what we know about ATP hydrolysis and what we do not know about ATP synthesis

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.     

ATP-induced ATP release from astrocytes

Propagation of interastrocyte Ca2+ waves is mediated by diffusion of extracellular adenosine triphosphate (ATP), and may require regenerative release of ATP. The ability of ATP to initiate release of intracellular ATP was assessed by labeling adenine nucleotide pools in astrocyte cultures with 14C-adenine. The 14C-purines released during exposure to ATP were then identified by thin-layer chromatography. ATP treatment caused a five-fold increase in release of 14C-ATP but not 14C-ADP or 14C-AMP, indicating selectivity for release of ATP. Other P2 receptor agonists also caused significant 14C-ATP release, and the P2 receptor antagonists suramin, reactive blue-2 and pyridoxalphosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) inhibited ATP-induced 14C-ATP release to varying degrees, suggesting the involvement of a P2 receptor. ATP-induced 14C-ATP release was not affected by chelation of intracellular Ca2+ with BAPTA-AM, or by blockers of Ca2+ release from intracellular stores or of extracellular Ca2+ influx, suggesting a Ca2+-independent response. ATP-induced 14C-ATP release was significantly inhibited by non-selective anion channel blockers but not by blockers of ATP-binding cassette proteins, gap junction hemichannels, or vesicular exocytosis. Release of adenine nucleotides induced by 0 Ca2+ was, in contrast, not selective for ATP, and was susceptible to inhibition by gap junction blockers. These findings indicate that astrocytes are capable of ATP-induced ATP release and support a role for regenerative ATP release in glial Ca2+ wave propagation.     

NMR observability of ATP: preferential depletion of cytosolic ATP during ischemia in perfused rat liver

The extent to which cellular metabolites are NMR observable is of fundamental importance in the interpretation of in vivo NMR studies. Analysis of ischemic rat liver shows that ATP resonances measured by 31P NMR decrease considerably faster than total tissue ATP measured in extracts. This discrepancy demonstrates that, in liver, ATP is not 100% observable. Furthermore, the data are consistent with the supposition that in situ mitochondrial ATP resonances are not normally observable by in vivo NMR techniques. The specificity of the NMR measurement for cytosolic ATP indicates that 31P NMR can be a valuable tool for the specific measurement of ATP in this compartment.     

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.     

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.     

Nucleotide-free kinesin hydrolyzes ATP with burst kinetics

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.     

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.     

ATP stimulates chemically sensitive and sensitizes mechanically sensitive afferents

We examined whether ATP stimulation of P2X purinoceptors would raise blood pressure in decerebrate cats. Femoral arterial injection of the P2X receptor agonist alpha,beta-methylene ATP into the blood supply of the triceps surae muscle induced a dose-dependent increase in arterial blood pressure. The maximal increase in mean arterial pressure (MAP) evoked by 0.1, 0.2, and 0.5 mM alpha,beta-methylene ATP (0.5 ml/min injection rate) was 6.2 +/- 2.5, 22.5 +/- 4.4, and 35.2 +/- 3.9 mmHg, respectively. The P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (2 mM ia) attenuated the increase in MAP elicited by intra-arterial alpha,beta-methylene ATP (0.5 mM), whereas the P2Y receptor antagonist reactive blue 2 (2 mM ia) did not affect the MAP response to alpha,beta-methylene ATP. In a second group of experiments, we tested the hypothesis that ATP acting through P2X receptors would sensitize muscle afferents and, thereby, augment the blood pressure response to muscle stretch. Two kilograms of muscle stretch evoked a 26.5 +/- 4.3 mmHg increase in MAP. This MAP response was enhanced when 2 mM ATP or 0.1 mM alpha,beta-methylene ATP (0.5 ml/min) was arterially infused 10 min before muscle stretch. Furthermore, this effect of ATP on the pressor response to stretch was attenuated by 2 mM pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (P < 0.05) but not by the P1 purinoceptor antagonist 8-(p-sulfophenyl)-theophylline (2 mM). These data indicate that activation of ATP-sensitive P2X receptors evokes a skeletal muscle afferent-mediated pressor response and that ATP at relatively low doses enhances the muscle pressor response to stretch via engagement of P2X receptors.     

Synthesis of free ATP from membrane-bound ATP in chromatophores of Rhodospirillum rubrum

Chromatophores of Rhodospirillum rubrum were preincubated with ³²P_i in the absence of added nucleotides. Particles and reaction mixture were then separated by sucrose density gradient centrifugation. The labeled chromatophores thus obtained esterify ³²P_i into acid-soluble ATP (ATP_as) on the addition of ADP in the dark. Additional firmly bound ATP (ATP_fb) can be liberated on sodium dodecylsulfate treatment. Coinciding with the formation of acid-soluble ATP there is a decrease in the amount of firmly bound ATP. The isotopic concentration experiments in which labeled chromatophores were incubated with carrier-free ³²P_i and ADP in the dark, show that ATP_as might arise from ATP_fb not by a direct γ-phosphate transfer but by an esterification of the added ADP and free phosphate with a concomitant hydrolysis of the ATP_fb. On this basis we have proposed a new working hypothesis for the last step of electron transport-linked phosphorylations. It includes the following reactions: + P*_i → P* (i.e., ATP_fb) P* + ADP + P**_i → ATP**_as + P*_i

The hypothesis is compatible with the concept of conformational energy conservation.     

Phosphoenzyme formation from ATP in the ATPase of sarcoplasmic reticulum. Effect of KCl or ATP and slow dissociation of ATP from precursor enzyme-ATP complex

The ATP-dependent phosphoenzyme formation and its reversal were studied at 0 degrees C and pH 7.0 in the ATPase of sarcoplasmic reticulum. Addition of KCl or several other salts (approximately 100 mM) decreased the maximum rate of ADP-induced dephosphorylation of phosphoenzyme as well as the apparent affinity of the phosphoenzyme toward ADP. High ATP had a similar effect on the latter, whereas it had little effect on the former. In contrast, high KCl or a considerable change in the ionic strength had little effect on the initial rate of phosphoenzyme formation at saturating ATP concentrations. During steady state phosphorylation at 1.0 mM MgCl2 and 5.0 mM CaCl2 in the absence of added KCl, a significant amount of [gamma-32P]ATP remained bound to the enzyme even when the enzyme concentration was much in excess over that of [gamma-32P]ATP. Evidence is presented that this enzyme-ATP complex represents a precursor to the phosphoenzyme. ATP dissociated slowly (0.20 s-1) from this enzyme-ATP complex and addition of high KCl or other salts accelerated its dissociation. In contrast, when the enzyme was complexed with adenyl-5'-yl (beta, gamma-methylene)diphosphonate in the absence of added KCl under these conditions, dissociation of the nucleotide from the complex as estimated in the displacement experiment with [gamma-32P]ATP, was found to be much faster than that of ATP.     

ATP release through pannexon channels

Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut.     

Kinetic modeling of ATP synthesis by ATP synthase and its mechanistic implications

Based on the torsional mechanism of ATP synthesis by ATP synthase, a kinetic scheme has been developed in this work. The scheme considers adenine nucleotide transport, binding of substrates ADP and P(i), unbinding of product ATP, and ATP synthesis. This kinetic scheme has been analyzed mathematically, and a kinetic model has been obtained to explain the experimentally observed hyperbolic Michaellian dependence of the rate of ATP synthesis on the ADP concentration by ATP synthase under physiological steady-state operating conditions. The principal results of the kinetic model have been compared with the experimental data and an estimate of the enzymological kinetic parameters V(max), K(M), and K(I) has been determined. Mechanistic implications arising from further analysis of the kinetic model have been discussed. These biological implications provide deep insight into the sequence of events leading to ATP synthesis.     

Localization of the ATP binding site on alpha-tubulin

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.     

胞外ATP的作用,来源和命运

胞外ＡＴＰ（三磷酸腺苷）浓度的改变,影响许多重要的生理功能,诸如神经递质作用、上板凝聚、篾这紧张、心脏功能肉收缩等。胞外ＡＴＰ作为递质直接影响神经效应器接点和／调制其它神经递质以及通过第二信使调节细胞活动,都是由膜上不同的ＡＴＰ受体或激活的离子通道介导的。本文从胞外ＡＴＰ的递质作用的发现与确证,胞外ＡＴＰ的来源、ＡＴＰ的受体、ＡＴＰ介导的反应及包外ＡＴＰ的降解等几方面对胞外ＡＴＰ研究的进展作一综述