Comparison of the self-association properties of the 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP). Further evidence for ionic interactions in the highly stable dimeric [H2(ATP)]2(4-) stack

The concentration dependence of the chemical shifts for the hydrogens H-2, H-8 and H-1' of ITP and for H-8 and H-1' of GTP has been measured in D2O at 25 degrees C under several degrees of protonation in the pD range 1.2-8.4. For reasons of comparison, inosine and guanosine have been included in the study The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 1.5 M-1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self-association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3-/D2(ITP)2- are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3-/D2(GTP)2-; in all these pairs the K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of the protonation degree on the self-association properties of adenosine 5'-triphosphate (ATP)

The concentration dependence of the chemical shifts for the protons H-2, H-8 and H-1' of ATP has been measured in D2O at 27 degrees C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD greater than 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD less than 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP4- (K = 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)-2- (6.0 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K approximately less than 17 M-1) (for reasons of comparison all constants are expressed in the isodesmic model). These results are compared with previous data for adenosine [Ado (K = 15 M-1) greater than 1:1 ratio of Ado/D(Ado)+ (6.0 M-1) greater than D(Ado)+ (0.9 M-1)] and AMP [AMP2- (K = 2.1 M-1) less than D(AMP)- (3.4 M-1) less than 1:1 ratio of D(AMP)-/D2(AMP) +/- (5.6 M-1) greater than D2(AMP) +/- (approximately equal to 2 M-1) greater than D3(AMP)+ (K less than or equal to 1 M-1)] to facilitate the interpretation of the results for the ATP systems. Stack formation of H2(ATP)2- is clearly favored by additional ionic interactions; this is confirmed by measuring via potentiometric pH titrations the acidity constants of H2(ATP)2- in solutions containing different concentrations of ATP. It is suggested that in the [H2(ATP)]4-(2) dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H+(N-1) site of one H2(ATP)2- and the gamma-P(OH)(O)-2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H2(ATP)4-(2) dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated.     

Self-association and protonation of adenosine 5'-monophosphate in comparison with its 2'- and 3'-analogues and tubercidin 5'-monophosphate (7-deaza-AMP)

The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1' for 2'-, 3'- and 5'-AMP2- and of the protons H-2, H-7, H-8 and H-1' for tubercidin 5'-monophosphate (= 7-deaza-AMP2-; TuMP2-) has been measured in D2O at 27 degrees C to elucidate the self-association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non-cooperative stacking; the association constants for all four NMPs are very similar: K approximately 2 M-1. These 1H-NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2- species exist predominately in the anti conformation. Comparison of the shift data for 5'-TuMP and 5'-AMP shows that no hydrogen bonding between N-7 and -PO3H- occurs; hence, the previously observed and confirmed 'wrongway' chemical shift [Martin, R. B. (1985) Acc. Chem. Res 18, 32] connected with the deprotonation of the -PO3H- group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N-7. From the dependence between the chemical shift and the pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D-ribose 5'-monophosphate. The measurements also allow an estimation of the first acidity constant of H3(5'-AMP)+ (pKDD3(AMP) = 0.9 and pKHH3(AMP) = 0.4). The values for pKHH2(NMP) and pKHH(NMP) were also determined from potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N-7 in the base moiety influence somewhat the acid-base properties of the mentioned NMPs. Measurements with 5'-AMP in 50% (v/v) aqueous dioxane show that lowering of the solvent polarity facilitates removal of the proton from the H+(N-1) site while the -PO2-3 group becomes more basic; this increases the pH range in which the monoprotonated H(5'-AMP)- species is stable and which is now also extended into the physiological pH region. Some consequences of this observation for biological systems are indicated.     

Self-association of adenosine 5'-monophosphate (5'-AMP) as a function of pH and in comparison with adenosine, 2'-AMP and 3'-AMP

The concentration dependence of the chemical shifts for protons H-2, H-8, and H-1' of adenosine (Ado), 2'-AMP, 3'-AMP and 5'-AMP was measured in D2O at 27 degrees C under several degrees of protonation. All results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for Ado decrease with increasing protonation: Ado (K = 15 M-1) greater than D(Ado)+/Ado (6.0 M-1) greater than D(Ado)+ (0.9 M-1). In contrast, a maximum is observed with 5'-AMP: 5'-AMP2- (K = 2.1 M-1) less than D(5'-AMP)- (3.4 M-1) less than D2(5'-AMP) +/- /D(5'-AMP)- (5.6 M-1) greater than D2(5'-AMP) +/- (approximately 2 M-1) greater than D3(5'-AMP)+ (less than or equal to 1 M-1). Self-stacking is most pronounced here if 50% of the adenine residues are protonated at N-1; complete base protonation reduces the stacking tendency drastically. Comparing the self-association of 2'-, 3'- and 5'-AMP shows that there is no influence of the phosphate-group position in the 2-fold negatively charged species, i.e., K congruent to 2 M-1 for all three AMP2- species. More importantly, there is also no significant influence observed if the stacking tendency of the three D2(AMP) +/- /D(AMP)-1:1 mixtures is compared (K congruent to 6-7 M-1); moreover, the measured association constants are within experimental error identical with the constant determined for D(Ado)+/Ado (K = 6.0 M-1). This indicates that any coulombic contribution between the -PO3(H)- group and the H+ (N-1) unit of the adenine residue to the stability of the mentioned stacks in D2O is small. However, experiments in 50% (v/v) dioxane-D8/D2O with the D2(5'-AMP) +/- /D(5'-AMP)- 1:1 system reveal, despite its low solubility, that coulombic interactions contribute to the self-association in an environment with a reduced polarity (compared to that of water). The implications of these observations for biological systems are briefly indicated.     

Self-association of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) and promotion by metal ions

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP4-) has been measured in D2O at 27 degrees C to elucidate the self-association. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 1.9 +/- 0.2 M-1, is only slightly larger than the value for ATP4-, K = 1.3 +/- 0.2 M-1. The self-stacking tendency of epsilon-ATP4- is promoted by a factor of about 4 by (1:1) coordination of Mg2+ to the phosphate moieties, which probably links these together and also neutralizes part of the negative charge; Zn2+ is only about half as effective as Mg2+ in promoting the self-association. This result contrasts with the self-stacking properties of Mg(ATP)2- and Zn(ATP)2-, Zn2+ being considerably more effective in a 1:1 ATP system. It is assumed that due to the enhanced affinity of the N-6/N-7 site of the epsilon-adenine moiety towards Zn2+ repulsion of the bases occurs resulting thus in a lower stacking tendency; in addition, the simple isodesmic model is no longer applicable to the Zn(epsilon-ATP)2- system: to explain the experimental data, the formation of an intermolecular metal ion bridge in the dimeric stacks is proposed. The experimental conditions required for studies of the properties of monomeric epsilon-ATP systems are described. Care should be exercised in employing epsilon-ATP as a probe for ATP.     

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.     

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

The effect of lead ions on the energy metabolism of human erythrocytes in vitro

The aim of this work was to evaluate the influence of chronic exposure to lead ions on the parameters of energetic status of human erythrocytes in vitro. Umbilical cord erythrocytes were incubated with lead acetate at final lead ion concentrations ranging from 10 to 200 microg/dl. ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, NAD and NADP concentrations in erythrocytes were determined using HPLC. Scanning electron micrographs of erythrocytes were taken. The mean concentrations of ATP, GTP, NAD and NADP, and mean values of adenylate energy charge (AEC) and GEC in cells incubated at the presence of lead ions were significantly lower after 20 h of incubation. Concentrations of purine degradation products (Ado, Guo, Ino) and Hyp were significantly higher. It is suggested that lead ions affect the energy metabolism of erythrocytes. Morphological changes in erythrocytes correspond to the increase of lead ions in the incubation mixture and to the decrease of ATP concentration in erythrocytes. A decrease in NAD and ATP concentration in erythrocytes could be a sensitive indicator of energy process disturbance, useful in monitoring in case of chronic lead exposure.     

Free metal ion depletion by "Good's" buffers. III. N-(2-acetamido)iminodiacetic acid, 2:1 complexes with zinc(II), cobalt(II), nickel(II), and copper(II); amide deprotonation by Zn(II), Co(II), and Cu(II)

Potentiometric, visible, infrared, electron spin, and nuclear magnetic resonance studies of the complexation of N-(2-acetamido)iminodiacetic acid (H2ADA) by Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) are reported. Ca(II) and Mg(II) were found not to form 2:1 ADA2- to M(II) complexes, while Mn(II), Cu(II), Ni(II), Zn(II), and Co(II) did form 2:1 metal chelates at or below physiological pH values. Co(II) and Zn(II), but not Cu(II), were found to induce stepwise deprotonation of the amide groups to form [M(H-1ADA)4-(2)]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed on the basis of various spectral data.     

Enhancement of glutathione production in a coupled system of adenosine deaminase-deficient recombinant Escherichia coli and Saccharomyces cerevisiae

Adenosine triphosphate (ATP) is necessary in the enzymatic production of glutathione (GSH). Our aim was to improve GSH production by increasing the efficiency of ATP regeneration in a coupled system. Previous results suggested that low GSH production in the coupled system is due to the irreversible transformation of adenosine (Ado) to hypoxanthine (Hx) via inosine (Ino) pathway in Escherichia coli JM109 (pBV03). In this study, to block the transformation of Ado into Hx and enhance GSH production, a coupled ATP regeneration system was constructed with adenosine deaminase-deficient recombinant E. coli JW1615 (pBV03) and Saccharomyces cerevisiae WSH2. GSH production was improved (2.94-fold of the control), and ATP regeneration reaction was established in the coupled system. The results are applicable to ATP regeneration in other microbial processes.     

Specific enzymatic determination of adenosine triphosphate

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).     

Specific enzymatic determinations of adenosine triphosphate.

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).     

Mathematical model of nucleotide regulation on airway epithelia. Implications for airway homeostasis

In the airways, adenine nucleotides support a complex signaling network mediating host defenses. Released by the epithelium into the airway surface liquid (ASL) layer, they regulate mucus clearance through P2 (ATP) receptors, and following surface metabolism through P1 (adenosine; Ado) receptors. The complexity of ASL nucleotide regulation provides an ideal subject for biochemical network modeling. A mathematical model was developed to integrate nucleotide release, the ectoenzymes supporting the dephosphorylation of ATP into Ado, Ado deamination into inosine (Ino), and nucleoside uptake. The model also includes ecto-adenylate kinase activity and feed-forward inhibition of Ado production by ATP and ADP. The parameters were optimized by fitting the model to experimental data for the steady-state and transient concentration profiles generated by adding ATP to polarized primary cultures of human bronchial epithelial (HBE) cells. The model captures major aspects of ATP and Ado regulation, including their >4-fold increase in concentration induced by mechanical stress mimicking normal breathing. The model also confirmed the independence of steady-state nucleotide concentrations on the ASL volume, an important regulator of airway clearance. An interactive approach between simulations and assays revealed that feed-forward inhibition is mediated by selective inhibition of ecto-5'-nucleotidase. Importantly, the model identifies ecto-adenylate kinase as a key regulator of ASL ATP and proposes novel strategies for the treatment of airway diseases characterized by impaired nucleotide-mediated clearance. These new insights into the biochemical processes supporting ASL nucleotide regulation illustrate the potential of this mathematical model for fundamental and clinical research.     

Nucleoside triphosphate specificity of tubulin

We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

Undifferentiated HL60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis

We have recently characterised the presence of a Ca2(+)-mobilising receptor for ATP which stimulates exocytosis in differentiated HL60 cells. Here we demonstrate that the undifferentiated HL60 cells also respond to extracellular ATP by stimulating an increase in inositol phosphates and exocytosis. Of the nucleotides (ATP, UTP, ITP, ATP gamma S, AppNHp, XTP, CTP, GTP, 8-Br-ATP and GTP gamma S) that were active in stimulating inositol phosphate formation, only UTP, ATP, ITP, ATP gamma S and AppNHp were active in stimulating secretion. On differentiation, the extent of secretion due to the purinergic agonists ATP, ITP, ATP gamma S and AppNHp remained unchanged whilst secretion due to UTP, a pyrimidine, was substantially increased. These results indicate that the effect of ATP and UTP may be mediated via separate purinergic and pyrimidinergic receptors, respectively.     

Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles

Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762-3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.     

Studies of the kinetics of the isolated mitochondrial ATPase using dinitrophenol as a probe

1. Dinitrophenol and maleate anions increase VATP on the 'washed', isolated, mitochondrial ATPase. Hydrolyses of iso-GTP and 2'-deoxy ATP are also stimulated, while hydrolyses of other nucleoside triphosphates (ITP, GTP etc.) are not. 2. Preincubation with ATP, iso-GTP or 2'-deoxy ATP results in a metastable enzyme form with a raised V and a reduced Km. Dinitrophenol stimulates both ATP and ITP hydrolyses by this form. 3. The Arrhenius plot of ATP (but not ITP) hydrolysis by the isolated ATPase shows a break at about 18 degrees C, apparently because the rate limiting step of hydrolysis changes as the temperature rises. 4. Adenylyl beta, gamma-imidodiphosphate (AdoPP[NH]P) inhibits ITP hydrolysis in a pseudofirst order reaction. Its binding is competitive with ITP. If the enzyme is preincubated with ATP, the rate of AdoPP[NH]P binding increases. It is concluded that AdoPP[NH]P inhibits by binding to the hydrolytic site of the enzyme. 5. We conclude that ATP hydrolysis is limited by diphosphate release and ITP hydrolysis by bond splitting. Energy release during ATP hydrolysis is maximal at the ATP binding step, and during ITP hydrolysis at bond splitting.     

Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits.