On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.2 in the presence of 0.15 M K+ and 25 degrees C. We further show that hydroxyquinolines are not satisfactory indicators for optical determination of the Mg2+-nucleotide dissociation constant. From our determinations a low value of free Mg2+ (less than 1 mM) is established for all of the tissues studied, including perfused heart muscle, contrary to a recent report in the literature. Saturating human erythrocytes with Mg2+ results in an alpha- and beta-phosphorus resonance separation for intracellular ATP that is indistinguishable from that observed in a noncellular MgATP control under similar conditions, showing that MgATP resonances in this cell are unaffected by the cellular environment.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites

31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.     

Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.     

Competition between Li+ and Mg2+ for ATP in human erythrocytes. A 31P NMR and optical spectroscopy study

We have investigated the influence of Li+ on free intracellular Mg2+ concentration in human erythrocytes by 31P NMR and optical absorbance spectroscopies. In red cells loaded with 3 mM intracellular Li+, the chemical shift separation between the alpha- and beta-phosphate resonances of MgATP2- was approx. 0.9 ppm larger than that observed in Li+-free red cells. By analyzing the interaction of each red cell component with Mg2+ and Li+, we found that Mg2+ is displaced in part from MgATP2- upon addition of Li+ and that the released Mg2+ is bound to the red cell membrane causing an overall decrease in free intracellular Mg2+ concentration.     

A 31P-NMR study of the mechanism of and salt effect on Mg2(+)-ATP exchange

The study of exchange between free and metal-bound ligands by NMR methods is discussed with reference to differentiation between unimolecular dissociation of the metal-bound complex and biomolecular exchange of metal ion between two ligands. This is applied to exchange of ATP4- between the free and Mg2(+)-bound states and problems of interpretation in the presence of a strong kinetic salt effect are discussed. Contrary to a previous report, exchange is inferred to occur mainly via unimolecular dissociation of MgATP2- over a range of temperatures, concentrations, and pH values, which include those expected in vivo. For the model system Mg2(+)-tripolyphosphate, an activation energy of 52 +/- 5 kJ.mol-1, inferred to be that for dissociation of MgTPPH2-, is found for the exchange process.     

Estimation of Cytoplasmic Free Mg2+ Levels and Phosphorylation Potentials in Mung Bean Root Tips by In Vivo 31P NMR Spectroscopy

The cytoplasmic [MgATP]/[ATP]_free ratios, free Mg²⁺ concentrations,and phosphorylation potentials in mung bean [Vigna mungo (L.)Hepper] root tip cells were investigated by ³¹P nuclear magneticresonance spectroscopy. ³¹P NMR spectra show well defined peaksdue to G6P, cytoplasmic P_i, vacuolar P_i, ATP, UDP-glucose andnicotinamide adenine nucleotides. The concentrations of phosphorusmetabolites were determined from quantitative ³¹P NMR spectra.The [MgATP]/[ATP]_free ratio was 9.45. Accordingly, about 90%of the cytoplasmic ATP was complexed to Mg²⁺. Utilizing thedissociation constant (K_d) determined for MgATP, the cytoplasmicfree Mg²⁺ concentration was estimated to be 0.4mM. The NMR-derivedphosphorylation potential, [ATP]/([ADP][P_i]), was 960 M^-1. Thesodium azide treatment decreased the [ATP]/[ADP] ratio and thephosphorylation potential, and increased the [Mg²⁺]^free. Metabolicinhibition may have been enhanced by an increase in [Mg^2+freeand a decrease in the free energy change for ATP hydrolysis,which resulted due to a decrease in the ATP level. ¹Present address: National Food Research Institute, TsukubaCity, Ibaraki 305, Japan. (Received February 8, 1988; Accepted June 1, 1988)     

The roles of magnesium ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.     

Further characterization of nucleotide binding sites on chloroplast coupling factor one

The solubilized coupling factor from spinach chloroplasts (CF1) contains one nondissociable ADP/CF1 which exchanges slowly with medium ADP in the presence of Ca2+, Mg2+, or EDTA; medium ATP also exchanges in the presence of Ca2+ or EDTA, but it is hydrolyzed, and only ADP is found bound to CF1. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CF1 and heat-activated CF1 bind one ATP/CF1, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]-propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the beta subunit of CF1. Equilibrium binding measurements indicate that CF1 has two identical binding sites for ADP with a dissociation constant of 3.9 microM (in addition to the nondissociable ADP site). When MgATP is bound to CF1, one ADP binding site with a dissociation constant of 2.9 microM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 microM. Reaction of CF1 with the photoaffinity label 3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of alpha and beta subunits. No additional binding sites with dissociation constants less than 200 micro M are observed for MgATP with latent CF1 and for CaADP with heat-activated CF1. Thus, three distinct nucleotide binding sites can be identified on CF1, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.     

The alpha beta-methylene analogues of ADP and ATP act as substrates for creatine kinase. delta G0 for this reaction and for the hydrolysis of the alpha beta-methylene analogue of ATP

The alpha beta-methylene analogues of ATP and ADP, [alpha beta CH2]ATP and [alpha beta CH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10(-5)-times lower than that with normal ATP. The affinities of the analogues (especially [alpha beta CH2]ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25 degrees C, measured using 31P NMR, for the reaction Mg[alpha beta CH2]ATP + creatine in equilibrium Mg[alpha beta CH2]ADP + phosphocreatine + H+ is 2.2 X 10(-12) M compared with a value of 2.5 X 10(-10) M for the same reaction with the normal substrates, corresponding to a difference in delta G0 values of 11.7 kJ X mol-1. It follows that delta G0 for the hydrolysis of the terminal phosphate group of Mg[alpha beta CH2]ATP is less favourable by 11.7 kJ X mol-1 than that for MgATP.     

The pre-steady state and steady-state kinetics of the ATPase activity of mitochondrial F1

1. The lag time before maximum velocity of ATP hydrolysis is reached upon mixing ATP with F1 is much greater than can be explained by a simple Michaelis-Menten mechanism, and must be due to an activation reaction. The lag time is dependent on the concentration of MgATP (half-maximal at 30 microM) and is equal to 30 ms at infinite MgATP concentration. The initial rate of hydrolysis by nucleotide-depleted F1 is much greater than with normal F1. It is tentatively suggested that the activation reaction with normal preparations is due to replacement of firmly bound ADP by MaATP. 2. After the initial time lag, the reaction follows very closely first-order kinetics provided that the concentration of MgATP is much less than the Km and the reaction is completed within 2 s. This is not expected if the dissociation constant of the enzyme-MgADP complex, an intermediate in the enzymic reaction, is much lower than the Km as has been reported in the literature. The value of V/Km, calculated from the exponential decay, is very close to that calculated from independent measurements of V and Km. 3. The low values for Ki(ADP) reported in the literature were found to be due to a slow (in the order of seconds) formation of an inhibited MgADP-enzyme complex. Dissipation of this inhibited complex by ATP requires seconds. The dissociation constant of the MgADP-enzyme complex that is an intermediate in the enzyme reaction was found to be 150 microM. 4. ADP but not ATP becomes firmly bound to nucleotide-depleted F1 in the absence of Mg2+.     

Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP. Isotope partitioning of the dPFK:MgATP complex indicates a random addition of MgATP and F6P at low Mg(2+), with the rate of release of MgATP from the central E:MgATP:F6P complex 4-fold faster than the net rate constant for catalysis. This can be contrasted with the ordered addition of MgATP prior to F6P at high Mg(2+). The addition of fructose 2,6-bisphosphate (F26P(2)) has no effect on the mechanism at low Mg(2+), with the exception of a 4-fold increase in the affinity of the enzyme for F6P. At high Mg(2+), F26P(2) causes the kinetic mechanism to become random with respect to MgATP and F6P and with MgATP released from the central complex half as fast as the net rate constant for catalysis. The latter is in agreement with previous studies [Gibson, G. E., Harris, B. G., and Cook, P. F. (1996) Biochemistry 35, 5451-5457]. The overall effect of Mg(2+) is a decrease in the rate of release of MgATP from the E:MgATP:F6P complex, independent of the concentration of F26P(2).     

The H(+)-ATPase of the plasma membrane from yeast. Kinetics of ATP hydrolysis in native membranes, isolated and reconstituted enzymes

The H(+)-ATPase of the plasma membrane from Saccharomyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H(+)-ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis-Menten kinetics with Km = 0.2 mM MgATP (plasma membranes), Km = 2.4 mM MgATP (isolated enzyme) and Km = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstitution; the activation of the H(+)-ATPase by free Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme-Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme-substrate complex, Ks, increases from Ks = 30 microM (plasma membranes) to Ks = 90 microM (isolated enzyme). ATP binding to the H(+)-ATPase ('single turnover' conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second-order rate constant k1 = 2.6 x 10(4) M-1.s-1. From these observations it is concluded that the detergent used (Zwittergent TM 3-14) interacts reversibly with the H(+)-ATPase and that practically all H(+)-ATPase molecules are reconstituted into the liposomes with the ATP-binding site being directed to the outside of the vesicle.     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

Inactivation of H+,K+-ATPase by a K+-competitive photoaffinity inhibitor

A light-sensitive derivative, 2,3-dimethyl-8-[(4-azidophenyl)methoxy]imidazo[1,2-a]pyridine (DAZIP), of the drug 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (SCH 28080) has been synthesized and shown to be a K+-competitive inhibitor of gastric H+,K+-ATPase in the dark. The apparent dissociation constants calculated for DAZIP at pH 6.4 and 7.4 were 1.8 +/- 0.2 and 4.7 +/- 1.2 microM, respectively. Inhibition required binding of DAZIP to a luminal-facing site on the enzyme. Irradiation in the presence of DAZIP and 2 mM Mg2+ resulted in irreversible loss of ATPase activity that was more than 2-fold greater at pH 6.4 than at pH 7.4, showing the enhanced efficiency of covalent incorporation at the lower pH. Further photolyses were conducted at pH 6.4 in the presence of either 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), ATP and CDTA, or MgATP. The specificity of light-dependent, covalent insertion of DAZIP for the site of reversible inhibition was shown both by protection against photoinactivation given by K+ (the competing ligand) and by the observation that the amount of K+-protectable photoinactivation approached a maximum limiting value as a function of DAZIP concentration. The effectiveness of K+ in protecting against photoinactivation was 100-fold greater in the presence of ATP and CDTA than in the presence of either Mg2+ or CDTA and suggests the formation of a ternary complex of the apoenzyme with ATP and tightly bound K+. The dissociation constant for DAZIP (2 microM) calculated from photolyses in the presence of MgATP without added K+ agreed with the kinetic experiments and suggests that DAZIP inhibits turnover by binding to E.MgATP.     

31P nuclear magnetic resonance spectra of the thiophosphate analogues of adenine nucleotides; effects of pH and Mg2+ binding.

The 31P nuclear magnetic resonance (NMR) spectra of the adenine nucleotide thio analogues, AMPS, ADPalphaS, ADPbetaS, ATPalphaS, ATPbetaS, and ATPgammaS, have been studied. Of primary interest were the increased sensitivity of chemical shifts to protonation and to magnesium binding of these analogues compared with the corresponding effects on AMP, ADP, and ATP. The usefulness of the characteristic NMR parameters of the thio analogues as probes in enzymatic reactions is discussed. The A2 diastereoisomers of ADPalphaS and ATPalphaS and the A and B isomers of ATPbetaS were enzymatically synthesized and the diasterioisomers of ADPalphaS and ATPbetaS were distinguished by their 31P NMR parameters. The stereospecificity of the enzymatic reactions involving the thio analogues of nucleotides can therefore be determined by 31P NMR. The difficulty involved in assigning phosphate ligands of Mg in MgADP and MgATP and their analogues on the basis of the magnitude of chemical shift changes (deltadelta) induced by Mg binding upon each 31P is discussed in the context of the anomalies in deltadelta of each 31P observed upon protonation of the terminal phosphate group. It is concluded that chemical shift data cannot yield unequivocal information concerning the absolute structure of metal complexes of nucleotides but can be used to monitor changes in metal chelation, for example, upon binding to enzyme.     

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

Concentration of MgATP2- and other ions in solution. Calculation of the true concentrations of species present in mixtures of associating ions.

1. A simple method is described for calculating the free concentrations of all species in a mixture of several ionic components that associate at equilibrium to any extent and with any stoicheiometry. 2. It can readily be adapted to take account of species such as protons for which the free rather than the total concentrations are controlled. 3. It was applied to mixtures of adenine nucleotides, Mg2+ and other ions relevant to the study of glucokinase (EC 2.7.1.2), but the qualitative conclusions are not peculiar to this system. 4. ATP exists in a high and nearly constant proportion (about 80%) as MgATP2- in solutions in which the total MgCl2 concentration exceeds the total ATP concentration by 1-10 mM. 5. By contrast, the proportion of ATP present as MgATP2- varies greatly if the total MgCl2 and total ATP concentrations are varied in constant proportion.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K. 10. Modulation of catalytic properties by magnesium.

The association of phenylalanylptRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate. The macroscopic dissociation constants are 0.16 +/- 0.03 and 4.1 +/- mM. The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNA Phe has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants of 0.25 +/- 0.03 and 9.1 +/- 1.7 mM. At 2 mM Mg2+, a single dissociation constant of 5.0 +/- 0.5 mM is indicated. The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+. The Hill constant of the sigmoidal dependence is 3.6. The strengthening of the synergism is believed to reflect a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+. The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+. The effect is to decrease the value of the Michaelis-Menten constant of MgATP. Another effect is to increase the rate constant when tRNA Phe is present. At subsaturating [MgATP], the [Mg2+] dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNA Phe. The rate of the pyrophosphorolysis is enhanced by a decrease of the Michaelis-Menten constant of MgPPi. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme.