Analysis of the relationship between the reactivity of synthetic substrates and thrombin inhibitors and their structure

Kinetics of thrombin- and trypsin-catalyzed hydrolysis of diphenylacetyl-L-arginine esters was studied at pH 8.5 and 25 degrees C, and the antithrombin activity of in vitro synthesized compounds was examined. The anticlotting activity of arylsulphonyl-L-arginine methyl esters appeared to be higher than that of the derivatives of diphenyl arginine. Relations were found connecting polar (delta) and steric (Es) characteristics of substituent (R) in R-C6H4-SO2-Arg-OCH3 esters with their antithrombin activity in vitro or with efficiency of their thrombin-catalyzed hydrolysis. This gives supplementary possibilities for synthesis of new substrates and more potent thrombin inhibitors.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Pressure effects on substrate activation phenomena in the alpha-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.     

Dissociation and aggregation of lactic dehydrogenase by high hydrostatic pressure

As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and --SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Lüdemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90--98; (1978) Eur. J. Biochem. 86, 219--224]. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation (less than or equal to 10 min) at pH 6.0--8.5 and pressures of 0.3--1.0 kbar causes dissociation which is reversed at normal pressure. At 5 degrees C the activation volume is found to be delta V not equal to = -62 +/- 3cm3 . mol-1. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5 degress C is delta V not equal to = -97 +/- 3cm3 . mol-1. The results may be described by a reaction sequence comprisign pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive high-molecular-weight particles.     

Studies on the specificity of acetylaminoacyl-peptide hydrolase.

In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Maternally Inherited Regulation of P Elements in Drosophila Melanogaster Can Be Elicited by Two P Copies at Cytological Site 1a on the X Chromosome

Two P elements, inserted at the cytological site 1A on an X chromosome from an Drosophila melanogaster natural population (Lerik, USSR), were isolated by genetic methods to determine if they are sufficient to cause the P cytotype, the cellular condition that regulates the P family of transposable element. The resulting "Lerik P(1A)" line (abbreviated "Lk-P(1A)") carries only one P element in situ hybridization site but genomic Southern analysis indicates that this site contains two, probably full length, P copies separated by at least one EcoRI cleavage site. Because the Lk-P(1A) line shows some transposase activity, at least one of these two P elements is autonomous. The Lk-P(1A) line fully represses germline P element activity as judged by the GD sterility and snw hypermutability assays; this result shows that the P cytotype can be elicited by only two P element copies. However, the Lk-P(1A) line does not fully repress delta 2-3(99B) transposase activity in the soma, although it fully represses delta 2-3(99B) transposase activity in the germline (delta 2-3(99B) is an in vitro modified P element that produces a high level of transposase activity in both the germline and the soma). The germline regulatory properties of the Lk-P(1A) line are maternally transmitted, even when the delta 2-3(99B) element is used as the source of transposase. By contrast, the partial regulation of delta 2-3(99B) somatic activity is chromosomally inherited. These results suggest that the regulatory P elements of the Lk-P(1A) line are inserted near a germline-specific enhancer.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

The specificity of two proteinases that cleave adjacent to arginine, C1 esterase and acrosin, for peptide p-nitroanilide substrates

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.     

Al-ATP as an intracellular carrier of Al(III) ion.

1. Using 27Al and 31P NMR spectroscopy in conjunction with an Al lactate aqueous reagent at pH 7.2, Al complexes of ATP and of phospholipids were characterized in synthetic-aqueous and organic-phospholipid chemical systems and in the intact human red blood cell. 2. The observed 31P NMR chemical shifts of the Al-ATP complex in aqueous laboratory preparations or the intact human red blood cell were, respectively, alpha phosphate, -11.53 delta; beta phosphate, -22.65 delta; and gamma phosphate, -10.95 delta. 3. The observed complexed 27Al chemical shift was -2.22 delta. 4. The relative affinities for Al of the phospholipids determined from 31P NMR spectroscopic titrations were PA much greater than Cl much greater than PS greater than PG approximately equal to PI greater than PE plus approximately equal to PE much greater than SPH greater than PC.     

The thermodynamic essence of the reversible inactivation of Na+/K+-transporting ATPase by various digitalis derivatives is relaxation of enzyme conformational energy

This paper reports on the kinetic and thermodynamic parameters describing the interaction of selected digitalis derivatives with hog and guinea-pig cardiac (Na+ + K+)-ATPase (Na+/K+-transporting ATPase EC 3.6.1.37). 32 digitalis derivatives were characterized as to the values of the delta G0', delta G----not equal to, and delta G----not equal to quantities in their interaction with (Na+ + K+)-ATPase from hog cardiac muscle in the presence of ATP, Mg2+, Na+ and K+. Nine derivatives were additionally characterized as to the values of the delta H0', delta S0', delta H----not equal to, delta S----not equal to, delta H not equal to, and delta S not equal to quantities in their interaction with the hog enzyme promoted by ATP, Mg2+ and Na+ in the presence or absence of K+. The formation of the inhibitory complexes is in any case an endothermic, entropically driven process. The Gibbs energy barriers in the formation and dissociation of the complexes, delta G----not equal to and delta G----not equal to, are imposed by large, unfavourable delta H not equal to values. K+ decreases the delta G0' value by increasing the delta G----not equal to value more than the delta G----not equal to value. In comparison with hog (Na+ + K+)-ATPase, the interaction of three derivatives with guinea-pig cardiac enzyme in the presence of ATP, Mg2+, Na+ and K+ is characterized by lower delta G0' values caused by lower favourable delta S0' values, and is accompanied by lower delta G----not equal to values. The magnitude of the kinetic parameters and the characteristic of the thermodynamic quantities describing the interaction between various digitalis derivatives and (Na+ + K+)-ATPase, indicate the induction of substantial conformational changes in the enzyme protein. A large entropy gain in the enzyme protein, observed irrespective of enzyme origin and ligation, appears to be the common denominator of the inhibitory action of all digitalis derivatives studied, suggesting that the digitalis-elicited relaxation of high conformational energy (negentropy strain) of the enzyme protein is the thermodynamic essence of the reversible inactivation of (Na+ + K+)-ATPase.     

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.     

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.     

PKC delta phosphorylates p52ShcA at Ser29 to regulate ERK activation in response to H2O2

Both PKC delta and ShcA have been implicated in cell response to oxidative stress [Y. Hu, X. Wang, L. Zeng, D.Y. Cai, K. Sabapathy, S.P. Goff, E.J. Firpo, B. Li, Mol Biol Cell., 16 (2005) 3705-3718, B. Li, X. Wang, N. Rasheed, Y. Hu, S. Boast, T. Ishii, K. Nakayama, K.I. Nakayama, S.P., Goff, Genes Dev, 18 (2004) 1824-1837, E. Migliaccio, M. Giorgio, S. Mele, G. Pelicci, P. Reboldi, P.P. Pandolfi, L. Lanfrancone, P.G. Pelicci, Nature, 402 (1999) 309-313], yet their relationship in the response has not been studied. Here we report that PKC delta interacts with ShcA and this interaction is promoted by H(2)O(2). PKC delta and ShcA are also colocalized in the cytoplasm and displayed co-translocation in response to H(2)O(2). Activated PKC delta was able to phosphorylate ShcA at Ser29, as determined by mass spectrometry. These results suggest that ShcA, p66 and p52, are substrates that interact with PKC delta. This phosphorylation is critical in H(2)O(2) induced ERK activation as reconstitution with ShcA Ser29A failed to rescue ERK activation of ShcA-/- MEFs, while ShcA could. In line with this conclusion, inhibition of PKC delta with inhibitors is able to diminish H(2)O(2) induced ERK activation in MEFs. These results suggest that the interaction between PKC delta and ShcA and the phosphorylation of ShcA at Ser29 play important roles in ERK activation in cell response to H(2)O(2).     

Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.     

beta-Oxidation of the coenzyme A esters of elaidic, oleic, and stearic acids and their full-cycle intermediates by rat heart mitochondria.

beta-Oxidation rates for the CoA esters of elaidic, oleic and stearic acids and their full-cycle beta-oxidation intermediates and for the carnitine esters of oleic and elaidic acids were compared over a wide range of substrate and albumin concentrations in rat heart mitochondria. The esters of elaidic acid were oxidized at about half the rate of the oleic acid esters, while stearoyl-CoA was oxidized equally as rapid as oleoyl-CoA. The full-cycle beta-oxidation intermediates of elaidoyl-CoA (trans-16 : 1 delta 7, -14 : 1 delta 5, and -12 : 1 delta 3) were found to be oxidized at rates nearly equal to those for the corresponding intermediates of oleoyl-CoA. Therefore, after the first cycle of beta-oxidation, oleoyl-CoA and elaidoyl-CoA are oxidized at nearly equal rates. The activity of fatty acyl-CoA dehydrogenase was higher with elaidoyl-CoA and its full-cycle intermediates as substrates than with the corresponding cisisomers. It was concluded that the slower oxidation rate of elaidic acid is not due to slower oxidation of any of its full-cycle beta-oxidation intermediates, nor to slower activity of fatty acyl-CoA dehydrogenase, nor to outer mitochondrial carnitine acyltransferase. Possible explanations to account for the slower oxidation rate of elaidic acid are discussed.     

Monte Carlo simulations of supercoiling free energies for unknotted and trefoil knotted DNAs.

A new Monte Carlo (MC) algorithm is proposed for simulating inextensible circular chains with finite twisting and bending rigidity. This new algorithm samples the relevant Riemann volume elements in a uniform manner, when the constraining potential vanishes. Simulations are performed for filaments comprising 170 subunits, each containing approximately 28 bp, which corresponds to a DNA length of 4770 bp. The bending rigidity is chosen to yield a persistence length, P = 500 A, and the intersubunit potential is taken to be a hard-cylinder potential with diameter d = 50 A. This value of d yields the same second virial coefficient as the electrostatic potential obtained by numerical solution of the Poisson-Boltzmann equation for 150 mM salt. Simulations are performed for unknotted circles and also for trefoil knotted circles using two different values of the torsional rigidity, C = (2.0 and 3.0) x 10(-19) dyne cm2. In the case of unknotted circles, the simulated supercoiling free energy varies practically quadratically with linking difference delta l. The simulated twist energy parameter ET, its slope dET/dT, and the mean reduced writhe <w>/delta l for C = 3 x 10(-19) dyne cm2 all agree well with recent simulations for unknotted circles using the polygon-folding algorithm with identical P, d, and C. The simulated ET vs. delta l data for C = 2.0 x 10(-19) dyne cm2 agree rather well with recent experimental data for p30 delta DNA (4752 bp), for which the torsional rigidity, C = 2.07 x 10(-19) dyne cm2, was independently measured. The experimental data for p30 delta are enormously more likely to have arisen from C = 2.0 x 10(-19) than from C = 3.0 x 10(-19) dyne cm2. Serious problems with the reported experimental assessments of ET for pBR322 and their comparison with simulated data are noted. In the case of a trefoil knotted DNA, the simulated value, (ET)tre, exceeds that of the unknotted DNA, (ET)unk, by approximately equal to 1.40-fold at magnitude of delta l = 1.0, but declines to a plateau about 1.09-fold larger than (ET)unk when magnitude of delta l > or = 15. Although the predicted ratio, (ET)tre/(ET)unk approximately equal to 1.40, agrees fairly well with recent experimental measurements on a 5600-bp DNA, the individual measured ET values, like some of those reported for pBR322, are so large that they cannot be simulated using P = 500 A, d = 50 A, and any previous experimental estimate of C.     

Proton nuclear magnetic resonance studies on the kinetics of tryptic fragments of calmodulin upon calcium binding

The kinetics of the Ca2+-dependent conformational change of the tryptic fragments F12 (residues 1-75) and F34 (residues 78-148) of calmodulin were studied by 1H-NMR. Resonances of two phenylalanines, 16 (or 19) and 65 (or 68), N epsilon, N epsilon, N epsilon-trimethyllysine-115 and tyrosine-138 were examined by the saturation-transfer technique or computer-aided line-shape simulation to obtain the rate of the conformational exchange between the Ca2+-free form and the Ca2+-bound form. The rates for F12 and F34 in the presence of 0.2 M KCl at 22 degrees C were 300-500 s-1 and 3-10 s-1, respectively. Activation parameters are as follows: Delta H not equal to = 11(+/- 2) kcal X M-1 and delta S not equal to = -9(+/- 5) cal X K-1 X M-1 for F12, and delta H not equal to = 16(+/- 2) kcal X M-1 and delta S not equal to = -2(+/- 5) cal X K-1 X M-1 for F34. These kinetic data for the conformational exchange are in agreement with those of Ca2+ dissociation from the binding sites obtained by 43Ca-NMR and stopped-flow fluorescence studies.     

Effects of magnesium sulfate on an unfolding step of human cyanomet myoglobin.

The effects of magnesium sulfate (MgSO4) on an unfolding step of human cyanomet myoglobin (Mb) were examined for wild-type and three L-->A mutant Mbs. The unfolding was induced at acidic pH (3.6-4.5) with various concentrations of MgSO4 (0-2 M). The monophasic process was monitored by visible absorption spectroscopy. We observed quite nonlinear delta G not equal to-[MgSO4] relations for all the Mbs. delta G not equal to-[MgCl2] relations were also determined for a comparative study. Thermodynamic evaluation of the results indicated that an upward reflection of delta G not equal to-[MgSO4] relations in high [MgSO4] is caused by the strong Hofmeister effect of the salt. Results obtained for three mutants (L29A, L72A, and L104A) at pH 4.0 and 4.5 were consistent with our previous observation that the structure of the transition state is determined by the stability of Mb cores in the balance with the pH conditions of unfolding (T. Konno and I. Morishima. 1993. Biochim. Biophys. Acta. 1162:93-98).     

An ATP/2e-stoichiometry of 1 1/2 is thermodynamically possible for site 3 of oxidative phosphorylation

Free energy changes for ATP synthesis (delta GP) and 2e(-)-transfer across Site 3 (delta GR) were determined during oxidative phosphorylation by rat liver mitochondria. At static head, -delta GR/delta GP ranged narrowly between 1.55 and 1.59 with five different respiratory substrates. Thus, an ATP/2e- of 1 1/2 at Site 3 is thermodynamically possible with regards to overall reactants and products. Using nonequilibrium thermodynamics, phenomenological stoichiometries were close to 1 1/2 for all substrates suggesting that ATP/2e- at Site 3 is, in fact, 1 1/2. An ATP/2e- of 1 1/2 can only be possible if H+/O is 4 for cytochrome oxidase.     

31P NMR of Mg-ATP in dilute solutions: complexation and exchange.

1. Monovalent-cation [(CH3)4N+, K(I), Na(I)] ATP, 1 mM in nucleotide, in aqueous solutions at pH 7.2, 24 degrees C, generates 2 different 31P NMR spectra, depending upon the salt content of the solution. At salt concentrations below 10 mM, the 31P NMR signals are chemically-shifted upfield (Na salt: alpha, -11.44 delta; beta, -22.91 delta; gamma, -8.36 delta) and the beta- and gamma-groups are broadened (at half-height: alpha, 3.5 Hz; beta, 9.6 Hz; gamma, 69 Hz). Above 10 mM salt, the signals are shifted downfield and are narrow (Na salt: alpha, -11.09 delta, 1.9 Hz; beta, -21.75 delta, 3.3 Hz; gamma, -6.30 delta, 3.9 Hz). 2. The Na-Mg-ATP complex, corresponding to the composition Na6Mg1ATP2, yields a single set of 31P resonances at concentrations of nucleotide of 100 mM, that upon dilution to 0.2 mM, resolve into 2 sets of ATP resonances characterized by low-field and high-field beta- and gamma-group resonance pairs. This set of ATP resonances, in contrast to the resonance set at 100 mM ATP, are broad (100 mM in ATP: alpha, -10.7 delta, 3.7 Hz; beta, -20.1 delta, 15 Hz; gamma, -5.7 delta, 7.3 Hz. 0.2 mM in ATP: alpha, -10.7 delta, 47 Hz; beta, -18.8 and -21.6 delta, 316 and 274 Hz; gamma, -5.5 and -8.7 delta, 460 and 374 Hz). 3. This new data, in combination with data derived from a survey of metal-ion-ATP studies, are interpreted in terms of ATP dimers, incorporating 2 molecules of ATP and 2 metal cations, that exist in water under the physiological conditions of neutral pH, high salt content [135 mM K(I)] and ATP concentrations in the range of 3 mM. 4. A compilation of 31P in vivo and ex vivo data compared to a reference Mg-ATP chemical shift vs Mg/ATP ratio plot indicates that ATP is not fully Mg-saturated in living systems and that 41% exists as the Mg(ATP)2 complex.