A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

An alkaline inorganic pyrophosphatase is found in association with isolated spinach chloroplast membranes. The enzyme is not removed from chloroplasts by repeated washings in an iso-osmotic medium. Suspension of the chloroplasts in hyper- or hypo-osmotic medium, however, results in the loss of pyrophosphatase activity in the chloroplasts. Fractionation of an isolated chloroplast suspension by differential centrifugation yields chloroplast fractions possessing high levels of alkaline pyrophosphatase activity but practically devoid of cytoplasmic acid pyrophosphatase.The alkaline pyrophosphatase exhibits a pH optimum of 8.2–8.5. In addition, there is an absolute requirement for Mg²⁺, with maximal rates of pyrophosphate hydrolysis occurring at $\text{Mg}{}^{\mathrm{2+}}\text{PP}{}_{\mathrm{i}}$ ratios greater than 2. From these findings the actual substrate for the enzyme is evidently Mg₂P₂O₇⁰ with pyrophosphate (P₂O₇^4?) acting as a potent inhibitor.The enzyme is inhibited by high concentrations of ATP (>3 mm), but increasing the concentration of Mg²⁺ effectively relieves this inhibition. At lower ATP concentrations, however, there is a stimulation of pyrophosphatase activity.The rate of hydrolysis of pyrophosphate by isolated chloroplasts is not affected by methylamine, 4′-deoxyphlorizin, and light. The possible role of this enzyme in photophosphorylation is discussed.     

Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca²⁺- and Mg²⁺-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca²⁺ overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H₂O₂) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H₂O₂ to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H₂O₂ under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H₂O₂ and NMM were dependent on free [Mg²⁺]_i; the inhibition was stronger when cells were perfused with higher free [Mg²⁺]_i solutions via pipette. In addition, TRPM7 current was not inhibited by H₂O₂ when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg²⁺]_i, which is sufficient for TRPM7 inhibition by H₂O₂ in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H₂O₂ just like the wild-type TRPM7 in a [Mg²⁺]_i- and [ATP]_i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg²⁺]_i elevation.     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Lysophosphatidylcholine-activated,vanadate-inhibited,Mg2+-ATPase from radish microsomes

An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg²⁺-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol P_i · min^?1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg²⁺ or some other divalent cations (CO²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Zn²⁺ in order of decreasing effectiveness); Ca²⁺ could not substitute for Mg²⁺ and was strongly inhibitory in its presence. K⁺, Rb⁺ and Na⁺ and also to a lesser extent NH₄⁺ and Li⁺ were significantly stimulatory, while the anions NO₃^?, H₂PO₄^?, Cl^? and SO₄^2? were ineffective. Orthovanadate, N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the K_m for ATP with that for Mg²⁺ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent K_m of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.     

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S_0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K_m value. The enzyme showed an absolute requirement for divalent cations (either Mg²⁺ or Mn²⁺), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg²⁺-activated enzyme to varying degrees (Ni²⁺ > Zn²⁺ > Cu²⁺ > Ca²⁺ > Ba²⁺). The differences in the kinetic responses of the enzyme to Mg²⁺ and Mn²⁺ are discussed.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266?×?10³ units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25?kDa. They showed temperature optima at 50°C and pH optima at 5.0.?km for SPases was reported to be 1.05?mM and 0.186?mM for guaiacol and H₂O₂ respectively. The V_max/K_m value for o-dianisidine was 449 while for H₂O₂ it was 5?×?10⁵. Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5???M) but ferulic acid inhibits its activity at a much lower concentration (0.02???M). Enzymes were stimulated by metal cations like Cu²⁺, Ca²⁺ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn²⁺ and Mg²⁺. Alanine stimulated the enzyme activity (up to 33%; at 0?C100???M) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13?C57% at 0?C100???M).     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Ribulose 1,5-bisphosphate carboxylase and oxygenase from Thiocapsa roseopersicina: activation and catalysis.

Ribulose 1,5-bisphosphate carboxylase/oxygenase purified from malate-grown Thiocapsa roseopersicina required Mg²⁺ for the activation of both carboxylase and oxygenase activities. Mg²⁺ was either not required or required at very low concentrations for catalysis by both enzyme activities. EDTA and dithiothreitol had no effect on ribulose 1,5-biphosphate oxygenase. The K_0.5 values with respect to Mg²⁺ for activation of the carboxylase and oxygenase activities were 8.4 and 2 mm, respectively. Ribulose 1,5-biphosphate carboxylase and oxygenase activities revealed differential sensitivities to 6-phosphogluconate. This ligand at 1 mm inhibited the carboxylase activity 30%, whereas the oxygenase activity was inhibited by 69%.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.     

Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca²⁺- and Mg²⁺-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K_m values of Ca²⁺ and Mg²⁺ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca²⁺- or Mg²⁺-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca²⁺ or Mg²⁺ or from two different enzymes are discussed.     

Influence of Certain Cations on Activity of Succinyl CoA Synthetase From Tobacco

Succinyl CoA synthetase from Nicotiana tabacum exhibited a requirement for univalent and divalent cations. Mn²⁺ replaced Mg²⁺ in the assay medium and Co²⁺ and Ca²⁺ partially replaced Mg²⁺. Addition of Zn²⁺ resulted in no enzyme activity. The enzyme was activated by univalent cations K⁺, Rb⁺, NH₄⁺, and Na⁺; Li⁺ showed little or no activation. Maximum enzyme activity varied significantly with potassium salts of different anions. Greatest activation was obtained with K₃PO₄ and, respectively, KCl, KNO₃, K₂SO₄ and KF exhibited steadily decreasing enzyme activation.     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by specific chemical modification of a lysine residue.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains nine lysine residues per subunit and can be inactivated by reagents specific for this amino acid. Pyridoxal-P reversibly inhibited the enzyme by about 70% by forming a Schiff base derivative with lysine. Reduction with NaBH₄ made this inactivation irreversible. Kinetic experiments indicated that the failure to inactivate the enzyme completely in a single treatment with pyridoxal-P reflects a reversible equilibrium between inactive Schiff base and a noncovalent complex. Modification of one lysine residue per subunit correlated with apparently total loss of activity. The rate of inactivation of the enzyme was decreased fourfold by saturating concentrations of ATP and was decreased at least 20-fold by formation of a quaternary complex of the enzyme with Mg²⁺, α,β-methylene ATP, and ribose-5-P. Trinitrobenzenesulfonate also irreversibly inactivated the enzyme, but this reagent was less specific in that the loss of activity corresponded to the modification of four to five lysine residues. These results suggest that an essential lysine is near the active site of Phosphoribosylpyrophosphate synthetase.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

The effect of hormones on metal and metal-ATP interactions with fat cell adenylate cyclase.

1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S_0.5(Mg²⁺) (concentration yielding 0.5 V_max) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its K_i(ATP^4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP^4? or Mg²⁺; its saturation with ATP^4? and citrate^3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in K_m and increase in V_max of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg²⁺ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg²⁺. The increase in the state of saturation of the enzyme with Mg²⁺ decreases the hormone-dependent effects on V_max and results in identical values of K_m (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl₂ saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl₂ show that the substitution of 5 mM MgCl₂ by 10 mM and 20 mM MgCl₂ increased the K_i(Ca²⁺) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its K_i(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl₂ exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its K_i(Ca²⁺), although only ACTH increased its K_i(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg²⁺ and Ca²⁺ because ATP^4? has stronger binding constants for Mg²⁺ and Ca²⁺ than ADP^3? and AMP^2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg²⁺, Ca²⁺, ATP^4? -sensitive adenylate cyclase.     

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.     

Mg2+-Dependent,cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from storage roots of red beet (Beta vulgaris L.) posess a Mg²⁺-dependent, alkaline pyrophosphatase (PPase) activity which is further stimulated by salts of monovalent cations. The requirement for Mg²⁺ is specific. Mn²⁺ and Zn²⁺ permitted only 20% and 12%, respectively, of the PPase activity obtained in the presence of Mg²⁺ while Ca²⁺, Co²⁺ and Cu²⁺ were ineffective. Stimulation of Mg²⁺-PPase activity by salts of certain monovalent cations was due to the cation and the order of effectiveness of the cations tested was K⁺=Rb⁺=NH ₄ ⁺ >Cs⁺. Salts of Li⁺ and Na⁺ inhibited Mg²⁺-PPase activity by 44% and 24%, respectively. KCl-stimulation of Mg²⁺-PPase activity was maximal with 60–100 mM KCl. There was a sigmoidal relationship between PPase activity and Mg²⁺ concentrations which resulted in markedly non-linear Lineweaver-Burk plots. At pH 8.0, the optimal [Mg²⁺]:[PP_i] ratio for both Mg²⁺-PPase and (Mg²⁺+KCl)-PPase activities was approximately 1:1, which probably indicates MgP₂O₇ ^2- is the true substrate.Abbreviations BSA bovine serum albumen - EDTA ethylenediamine tetra-acetic acid, disodium salt - MES 2-(N-morpholino)ethanesulphonic acid - Mg _T ²⁺ total magnesium - P_i inorganic phosphate - PPase inorganic pyrophosphatase - PP_i inorganic pyrophosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine