Measurement of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31P NMR and optical absorbance spectroscopy

To resolve a controversy in the literature concerning the affinity of Mg++ for ATP to be used in our noninvasive 31P NMR procedure for the determination of free Mg++ in living cells, we have reinvestigated the apparent dissociation constant of MgATP under physiologic ionic conditions and over the cellular range of ATP concentrations by a combination of NMR and optical absorbance techniques. The new combination method utilizes 31P NMR chemical shifts to determine the degree of Mg++ chelation of ATP in a solution containing free ATP and MgATP, and uses a properly calibrated indicator dye, antipyrylazo III, for optical measurement of free Mg++ in the same solution. The data yield an average value of 50 +/- 10 microM for the apparent dissociation constant of MgATP which indicates low levels of free Mg++ (less than 1 mM) in several different types of tissues, including perfused heart muscle, contrary to a recent report in the literature.     

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.     

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.     

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.     

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.     

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.     

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.     

Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy

The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.     

Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria

Null point titration techniques have been developed for measurements of cytosolic free Mg2+ in isolated cells and matrix free Mg2+ in isolated mitochondria using antipyrylazo III as a spectrophotometric Mg2+ indicator. A cytosolic free Mg2+ of 0.37 +/- 0.02 mM was obtained with hepatocytes. This represented about 6% of the total cytosolic magnesium content (activity coefficient of 5.8 X 10(-2). Nondiffusable Mg2+-binding sites in the cytosol were equal to 11.1 nmol/mg cell dry weight with an apparent dissociation constant of 0.71 mM and accounted for binding of 32% of the cytosolic magnesium. The null point method gave a value of 0.35 +/- 0.01 mM for the mitochondrial matrix free Mg2+ concentration (activity coefficient of 8.8 X 10(-3). Nondiffusable Mg2+ binding sites in the mitochondria were estimated at 25.7 nmol/mg mitochondrial protein with an apparent dissociation constant of 0.22 mM, compared with an apparent dissociation constant of 1.66 microM for bound calcium. These data demonstrate the absence of a significant gradient of free Mg2+ between the cytosolic and mitochondrial compartments. They also demonstrate a high ligand binding capacity for magnesium in both compartments with relatively low affinity resulting in a constant value for free Mg2+ when total cell magnesium is constant. This maintains a ratio between free Mg2+ and free Ca2+ of about 2000 in the cytosol and 100 in the mitochondria. The high concentration and low affinity of Mg2+ binding sites results in rather large changes of free Mg2+ with small variations in total cell magnesium. This is apparent in hepatocytes isolated from streptozotocin diabetic rats which had a decreased total magnesium content and a cytosolic free Mg2+ of 0.16 +/- 0.02 mM.     

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.     

Phosphorus-31 nuclear magnetic resonance studies on intact erythrocytes. Determination of intracellular pH and time course changes in phosphorus metabolites

A method to determine the intracellular pH of intact erythrocytes using phosphorus-31 nuclear magnetic resonance spectroscopy is described. Changes in phosphorus metabolites due to the alkalization of intracellular pH were also examined. The normal erythrocytes gave signals of phosphate groups corresponding to 2,3-bisphosphoglycerate, inorganic phosphate, ATP, and NAD. Among them, the separation between alpha and gamma peaks of ATP was shown to be a good indicator of the intracellular pH free from the perturbation caused by hemoglobin. This method enabled us to determine the intracellular pH of the erythrocytes without any pretreatment. The separation between alpha and gamma peaks of ATP was also dependent on the degree of complexation with Mg2+, and was consistent with approximately 80% of total ATP complexing with Mg2+ in the samples investigated here. The pKa value of ATP in the erythrocytes was estimated to be 6.1 at 23 degrees C, which is lower than the value of 6.5 obtained for the Mg2+-free ATP solution. In the alkalized erythrocytes, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were observed in addition to the metabolites found in the normal erythrocytes. Time course changes in these phosphorus metabolites were followed along with the intracellular pH monitored from ATP peaks.     

A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum.

Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude.     

19F nuclear magnetic resonance studies of free calcium in heart cells.

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.     

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.     

Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.     

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.     

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)     

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.     

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. II. Kinetic studies

The rate of ATP hydrolysis by the Neurospora plasma membrane [H+]-ATPase has been measured over a wide range of Mg2+ and ATP concentrations, and on the basis of the results, a kinetic model for the enzyme has been developed. The model includes the following three binding sites: 1) a catalytic site at which MgATP serves as the true substrate, with free ATP as a weak competitive inhibitor; 2) a high affinity site for free Mg2+, which serves to activate the enzyme with an apparent K1/2 (termed KMgA) of about 15 microM; and 3) a separate low affinity site at which Mg2+ causes mixed type inhibition, lowering the Vmax while raising the KS for MgATP at the catalytic site. The Ki for Mg2+ at the low affinity site (termed KMgI) is about 3.5 mM. The model satisfactorily explains the activity of the enzyme as Mg2+ and ATP are varied, separately and together, over a wide range. It can also account for the previously reported effects of Mg2+ and ATP on the inhibition of the Neurospora [H+]-ATPase by N-ethylmaleimide (Brooker, R. J., and Slayman, C. W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832).