A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg²⁺, Mn²⁺, Ca²⁺, ATP^4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP^4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K _m(MgATP)=1.0 mM,K _i(ATP^4–)=0.27 mM,K _m(MnATP)=0.07 mM, andK _i(CaATP)=0.015 mM. MgATP, MnATP, ATP^4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP^4– from MgATP. Ca²⁺ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK _0.5(Ca²⁺)=0.02 mM,K _0.5(Mn²⁺)=3.8 mM,K _0.5(Mg²⁺)=4.7 mM, and an order of activity Mn²⁺>Mg²⁺>Ca²⁺. The data indicate that Mn²⁺ and Mg²⁺ ions may compete for a regulatory site distinct from the active site and increaseV _m without changingK _m(MgATP),K _m(MnATP), orK _i(ATP^4–). The interactions of ATP^4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg²⁺ and Mn²⁺, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg²⁺ nor Mn²⁺, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP^4– and CaATP are involved in dead-end inhibition. For MgCl₂ saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg²⁺ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg²⁺.Abbreviations used: Me, divalent metal; Me_T (Mg_T or Mn_T), total Me (Me²⁺ and its complexes); ATP_T, total ATP (ATP^4– and its complexes).     

MOUSE NEUROBLASTOMA CELL ADENYLATE CYCLASE: REGULATION BY 2-CHLOROADENOSINE, PROSTAGLANDIN E1 AND THE CATIONS Mg2+, Ca2+ AND Mn

—Some basic kinetic properties of adenylate cyclase in cell free preparations of mouse neuroblastoma were investigated. Production of cAMP from ATP by the enzyme requires the presence of either Mg²⁺ or Mn²⁺ in addition to ATP. In the presence of Mg²⁺, the K_m for ATP is 120 ± 15 μM and the interaction of ATP and adenylate cyclase appears to be non-cooperative (Hill coefficient of 1). Magnesium ion concentrations in excess of the ATP concentration cause stimulation although similar excess concentrations of Mn²⁺ cause inhibition. Prostaglandin E₁ and 2-chloroadenosine activate the enzyme. The K_m of the cyclase for 2-chloroadenosine is 6 μm . Activation by 2-chloroadenosine leads to an increase in V_max but does not effect the K_m for ATP. At a fixed ATP concentration, the extent of activation caused by prostaglandin E₁ and 2-chloroadenosine is inversely related to the Mg²⁺ concentration. Calcium ion causes inhibition of adenylate cyclase from 0.1 to 4mM with a K_i of 5 ± 10^?4m . Ca²⁺ interaction with the enzyme in the absence or presence of either 2-chloroadenosine or prostaglandin E₁ appears cooperative (i.e. Hill coefficients of ?2). Ca²⁺ inhibition is non-competitive with respect to either ATP or 2-chloroadenosine but is progressively diminished by increasing Mn²⁺ concentrations. Divalent cation effects and activation by 2-chloroadenosine and prostaglandin E₁ of the neuroblastoma adenylate cyclase are compared with ion effects and hormone activation of the enzyme obtained from non-neuronal tissue.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Ca-stimulated, Mg-independent ATP hydrolysis and the high affinity Ca-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m = 0.25 μM, V_max = 24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties

Purified glutamine synthetase from pea seedlings was most active with Mg²⁺ as the metal activator, but Mn²⁺ and Co²⁺ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg²⁺ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co²⁺ also gave a sigmoidal saturation curve, but when Mn²⁺ was varied only slightly sigmoidal kinetics were seen. Addition of Mn²⁺, Ca²⁺, or Zn²⁺ at low concentrations sharply inhibited the Mg²⁺ -dependent activity, partially by shifting the pH optimum. Addition of Co²⁺ did not inhibit Mg²⁺-dependent activity. The nucleotide triphosphate specificity changed markedly when Co²⁺ or Mn²⁺ replaced Mg²⁺. Using the Mg²⁺-dependent assay, the Michaelis constant (Km) for NH₄⁺ was about 1.9 × 10⁻³ M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%.     

Modification of the kinetics and regulatory properties of bovine hepatic fructose 1,6-diphosphatase with pyridoxal 5'-phosphate

Treatment of purified fructose 1,6-diphosphatase from bovine liver (which is maximally active at neutral pH) with pyridoxal 5'-phosphate produces changes in the spectral, catalytic, and allosteric properties of the enzyme. After modification the Michaelis constants for fructose-1,6-diphosphate and Mg²⁺ are increased, and inhibition by AMP is decreased. Substrate inhibition is decreased, but not abolished; the curve is merely shifted toward higher substrate concentration. Fructose-1, 6-diphosphate protects against the increases in the K_m for fructose-1, 6-diphosphate and the K_m for Mg²⁺, and against the changes in substrate inhibition, but not against the changes in AMP inhibition. AMP protects against the changes in AMP inhibition and the increase in the K_m for magnesium, but does not prevent the changes in substrate inhibition or the increase in the K_m for fructose-1, 6-diphosphate. The pH curves in the modified enzyme are altered at high, but not at low, substrate concentration.     

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F₁F_o-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K_0.5=0.27 mM) and low (K_0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg²⁺ saturating condition (high affinity site, K_0.5=0.10 mM, and low affinity site, K_0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.     

Pyruvate carboxylase from rainbow trout liver

Summary A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K⁺ causes a decrease in the apparentK _m for HCO ₃ ^– . AcetylCoA activation shows positive cooperativity withK _a=0.072 mM andn _H=1.78 at pH 7.7, 2.5 mM free Mg²⁺, 100 mM K⁺, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK _m values for MgATP and HCO ₃ ^– and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a U-type response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.Abbreviations DTT dithiothreitol - PMSF phenylmethylsulfonylfluoride     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose hisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

A microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na⁺ and K⁺ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na⁺ and K⁺. At low Na⁺ concentration (below 5 mM), the apparent K_m decreases with increasing concentrations of K⁺ (1–20 mM). At 5 mM Na⁺, the K⁺ level does not change the apparent K_m, while at Na⁺ levels above 10 mM, the apparent K_m between enzyme and substrate increases with increasing concentration of K⁺. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na⁺ and K⁺. The Na⁺ binding is cooperative with different K_m values and Hill coefficients (n) in the presence of low and high concentration of K⁺. At low Na⁺ level (< 5 mM). a negative cooperativity exists for Na⁺ (n_Na < 1) which is more pronounced in the presence of high [K⁺]. When the concentration of Na⁺ is raised the negative cooperativity disappears and turns into a positive one (n_Na > 1). Only K⁺ binding in the presence of low [Na⁺] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K⁺ (n_K < 1 → n_K > 1). In the presence of [Na⁺] > 10 mM, the changes in n_k are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na⁺, K⁺ and MgATP. 4. In the presence of Na⁺ (< 10 mM), K⁺ is probably bound to two K sites, one of which translocates K⁺ through the membrane by an antiport Na⁺/K⁺ mechanism. This could be connected with an elevated K⁺ uptake in the presence of Na⁺ and could therefore explain some field properties of sugar beets.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Effects of magnesium ion and chelating agents on enzymatic production of ATP from adenine

Summary As reported previously, enzymatic production of ATP from adenine by resting cells of Brevibacterium ammoniagenes (Fujio and Furuya 1983) accumulated 13.0 mg of ATP · Na₂ · 3H₂O/ml, but ATP formation ceased within 6–8 h. Simultaneous addition of magnesium ion and phytic acid, a chelator of divalent cations, allowed ATP formation to continue longer, and 24.2 mg of ATP · Na₂ · 3H₂O/ml was accumulated in 10 h. However, ATP formation ceased thereafter.This second cessation was found to be caused by the lack of magnesium ion active as a co-factor (Mg^act). The Mg^act was tentatively taken as the difference between soluble magnesium ion (Mg^sol) and the ion chelated by an equimolar amount of ATP (Mg^ATP), namely Mg^act=Mg^sol-Mg^ATP. In order to provide Mg^act, sufficient phytic acid had to be added at the beginning of the reaction and magnesium ion was also added intermittently. Under these conditions ATP formation continued further, and the rate of ATP formation was increased; 37.0 mg of ATP · Na₂ · 3H₂O/ml was accumulated in 13 h.Since whole culture broth is preferable to frozen cells as a practical enzyme source, the conditions neccessary for use of whole culture broth of B. ammoniagenes were also investigated.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum

The (Ca²⁺-Mg²⁺)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg²⁺-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg²⁺-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K ₁=1 µM), EP.Ca₂ (K ₉=500 µM) and *EP (K ₇=20 µM), accelerating the release of P_i. The fact that each of these species has a different affinity for Mg²⁺-ATP allows a significant enhancement of the rate of P_i release to the medium at the different ranges of Mg²⁺-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg²⁺-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg²⁺-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3).     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.