The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

The effect of di- and trivalent cations on the phosphorylation of the Ca2+-ATPase in sarcoplasmic reticulum vesicles

The steady-state level of phosphorylated intermediate (EP) of (Mg2+ + Ca2+)-ATPase is influenced by magnesium and calcium concentration in the Ca2+-transporting system of sarcoplasmic reticulum vesicles. At micromolar [Ca2+], the level of EP is increased by Mg2+, depending on its concentration. The effect of Mg2+ is less pronounced at lower Ca2+ concentration. At low [Mg2+], the EP formation increases at millimolar concentrations of Ca2+, suggesting, in accordance with earlier results, that the substrate may also be CaATP instead of MgATP. LaCl3 (1 mM) enhanced the EP formation at low Mg2+ concentration. Surprisingly, 10 microM LaCl3 caused a marked decrease in EP formation at high [Mg2+] and had little or no effect on the level of EP at low Mg2+ concentration. The inducing effect of 1 mM LaCl3 on the EP formation at low [Mg2+] and the inhibitory effect of 10 microM LaCl3 at high Mg2+ concentration draw attention to the involvement of divalent cation-binding sites with different affinity in phosphorylation and to the particular role of Mg2+ in the EP formation and EP decomposition.     

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.     

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.     

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase.

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I

The mechanism of Ca2+ inhibition of carbamylphosphate synthetase I has been investigated using purified enzyme obtained from livers of rats fed a high protein diet. Binding of Mn2+ to the enzyme was measured by EPR techniques at pH 7.8, and Scatchard plots of the data indicated one Mn2+-binding site with a K'd of 13 microM. From competition studies between Mn2+ and Ca2+ or Mg2+ binding, values of 180 microM were obtained for K'd (Mg) and 193 microM for K'd (Ca). A nonlinear least squares curve fitting program was used to calculate the K'm for MgATP2- at the metal-nucleotide binding sites using a simplified rate equation of the enzyme reaction mechanism. Values of 140 and 2420 microM were obtained for K'm (MgATP) at the first and second sites, respectively, at pH 7.8, with a free Mg2+ of 1 mM and other substrates and activators present at saturating concentrations. Variations of the bicarbonate, N-acetylglutamate, and ammonia concentrations in the absence and presence of different amounts of total calcium, from which free Ca2+, free Mg2+, MgATP2-, and CaATP2- concentrations were calculated, permitted values for K'i (CaATP) to be obtained by graphic procedures. Mean values of 375 and 120 microM were obtained for K'i (CaATP) at the first and second sites, respectively. Using the above kinetic constants, a computer model of the enzyme reaction was constructed and tested using two further sets of kinetic data obtained by varying the concentrations of Mg2+, Ca2+, MgATP2-, and CaATP2-. Poor fits were obtained unless the formation of a mixed complex involving CaATP2- competition with MgATP2- at the second metal-nucleotide-binding site was incorporated into the rate equation. Nonlinear least squares curve fitting of both sets of experimental data gave a well determined value of 124 microM for this final CaATP2- inhibitory constant. Sensitivity tests for variation of the primary kinetic constants with the computer model showed that the inhibitory effect of free Ca2+ was weak and that the observed calcium inhibition of carbamylphosphate synthetase can be accounted for primarily by competitive interaction of CaATP2- at the second MgATP2- binding site. With 1 mM free Mg2+ and 5 mM MgATP2-, half-maximal inhibition of enzyme activity was obtained with 0.2 mM CaATP2-.     

Regulation of the F-ATPase from mitochondria of Vigna sinensis (L.) Savi cv. Pitiuba by spermine, spermidine, putrescine, Mg2+, Na+, and K+

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound of F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%. The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical. The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.     

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 an ionized Ca2+ concentration of approx. 1 microM. 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.     

Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.     

Interaction of sodium and potassium ions with Na+, K+-ATPase. III. Cooperative effect of ATP and Na+ on complete release of K+ from E2K

The effects of Na+ and ATP on the K+ binding to Na+, K+-ATPase were investigated by the centrifugation method with radioactive K+ in the absence of Mg2+. In the presence of 10 microM 43KCl, 0.6 and 10 mM Na+ decreased the amount of bound K+ to one-half and zero, respectively. On the other hand, 10 microM and 10 mM ATP decreased the amount of K+ to 60 and 25-40%, respectively. When the combined effect of ATP and Na+ was tested, 10 microM ATP decreased the Na+ concentration giving half-maximal inhibition of the K+ binding to one-third, showing synergistic inhibition by both ligands, though increase in ATP concentration seemed to depress the inhibitory effect of Na+. The synergistic inhibition by ATP and Na+ suggests that the release of K+ from E2K is not completed by the binding of ATP alone but is completed by the binding of Na+ in addition to ATP during the cycle of Na+, K+-dependent ATP-hydrolysis as well as ion-transport.     

Desensitization to Mg2+ of the phosphoenzyme in sarcoplasmic reticulum Ca2+-ATPase by the addition of a nonionic detergent and the restoration of sensitivity after its removal

Sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C12E8) and the kinetic properties of the Ca2+,Mg2+-dependent ATPase [EC 3.6.1.3] were studied. The following results were obtained: 1. SR ATPase solubilized in C12E8 retains high ability to form phosphoenzyme ([EP] = 4--5 mol/10(6) g protein) for at least two days in the presence of 5 mM Ca2+, 0.5 M KCl, and 20% glycerol at pH 7.55. 2. The ATPase activity was dependent on both Mg2+ and Ca2+. However, the rate of E32P decay after the addition of unlabeled ATP was independent of Mg2+. 3. Most of the EP formed in the absence of Mg2+ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5 mM Mg2+, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP. 4. The removal of C12E8 from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg2+ dependency of the EP decomposition. 5. These results strongly suggest that in the case of SR solubilized with a high concentration of C12E8 the decomposition of phosphoenzyme is Mg2+ independent and ATP is mainly hydrolyzed through Mg2+-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Energy interconversion in sarcoplasmic reticulum vesicles in the presence of Ca2+ and Sr2+ gradients

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle are able to accumulate Ca2+ or Sr2+ at the expense of ATP hydrolysis. Depending on the conditions used, vesicles loaded with Ca2+ can catalyze either an ATP in equilibrium Pi exchange or the synthesis of ATP from ADP and Pi. Both reactions are impaired in vesicles loaded with Sr2+. The Sr2+ concentration required for half-maximal ATPase activity increases from 2 microM to 60-70 microM when the Mg2+ concentration is raised from 0.5 to 50 mM. The enzyme is phosphorylated by ATP in the presence of Sr2+. The steady state level of phosphoenzyme varies depending on both the Sr2+ and Mg2+ concentrations in the medium. Phosphorylation of the enzyme by Pi is inhibited by both Ca2+ and Sr2+. In the presence of 2 and 20 mM Mg2+, half-maximal inhibition is attained in the presence of 4 and 8 microM Ca2+ or in the presence of 0.24 mM and more than 2 mM Sr2+, respectively. After the addition of Sr2+, the phosphoenzyme is cleaved with two different rate constants, 0.5-1.5 s-1 and 10-18 s-1. The fraction of phosphoenzyme cleaved at a slow rate is smaller the higher the Sr2+ concentration in the medium. Ca2+ inhibition of enzyme phosphorylation by Pi is overcome by the addition of ITP. This is not observed when Ca2+ is replaced by Sr2+.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.     

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.     

Adenylate cyclase in silkworm. Properties of the enzyme in pupal fat body.

Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+. The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP.