Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

Binary interactions of troponin subunits

The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.     

Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation

The mechanism of sarcoplasmic reticulum (SR) ATPase Mg2+-dependent phosphorylation from Pi was investigated in the presence of 15% v/v dimethyl sulfoxide at pH 6, 20 degrees C, and in the absence of potassium. Measurements of intrinsic fluorescence changes and of 32P-labeled phosphoprotein (*E-P) were in agreement, both at equilibrium and in transient situations. We found that the amount of phosphoenzyme present and its rate of formation depended solely on the concentration of the (Mg X Pi) complex. Up to 6 nmol of phosphate/mg of protein was covalently bound to the enzyme, implying almost complete phosphorylation. Oxygen exchange experiments were also performed in order to allow calculation of the absolute rate constant of *E-P hydrolysis to the noncovalent complex (0.8-1.0 s-1), which differs from the observed rate of enzyme dephosphorylation (0.3-0.5 s-1); in addition, they allowed calculation of the bimolecular rate constant of substrate binding (2-2.4 M-1 s-1). The results demonstrate that in the presence of dimethyl sulfoxide, phosphorylation occurs by the following simple mechanism: relatively slow binding of the neutral substrate (Mg X Pi), with poor affinity, followed by a thermodynamically favorable formation of the covalent bond between phosphate and the possibly hydrophobic active site. The interaction between magnesium and calcium-deprived SR vesicles was studied in the presence of 0-20% v/v dimethyl sulfoxide (or 0-30% v/v glycerol) at pH 7 and 20 degrees C. The presence of either solvent led to the disappearance of the two typical pH-dependent effects we previously characterized for magnesium: loss of the Mg2+-induced spectral shift of tryptophan fluorescence emission and loss of the biphasic pattern displayed by the intrinsic fluorescence rise after addition of calcium to Ca2+-deprived Mg2+-preincubated vesicles. In the absence of solvent, the interaction of magnesium with the calcium-deprived ATPase was also characterized from the point of view of phosphoenzyme formation from ATP or Pi at pH 7 in the absence of potassium: we found that calcium-independent phosphorylation was slower when phosphate was added to SR vesicles preincubated with magnesium that when magnesium was added to vesicles preincubated with phosphate, suggesting that preincubation with magnesium had depleted the phosphate-reactive conformation of the ATPase. A simple reaction scheme for phosphoenzyme formation is described: it implies that the (Mg X Pi) complex is a substrate for this reaction, whereas the Mg2+ itself acts as a pH-dependent, dimethyl sulfoxide sensitive inhibitor of full enzyme phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.     

Conformation and ion binding properties of peptides related to calcium binding domain III of bovine brain calmodulin

The conformational and ion binding properties of the sequences 93-104, 96-104, and 93-98 of domain III of bovine brain calmodulin (CaM) have been studied by CD and Tb3+-mediated fluorescence. In aqueous solution the interaction of all fragments with Ca2+ and Mg2+ ions is very weak and without any effect on the peptide conformation, which remains always random. In trifluoroethanol the interaction is very strong and the different fragments exhibit very distinct binding properties. In particular, the dodecapeptide fragment 93-104, and its N-terminal hexapeptide 98-104, bind calcium and magnesium with a very high binding constant (Kb greater than 10(5) M-1), undergoing a substantial conformational change. The structural rearrangement is particularly evident in the hexapeptide fragment, which tend to form a beta-bend. The C-terminal nonapeptide fragment 96-104 interacts with calcium and magnesium more weakly, and the binding process causes a decrease of ordered structure. These results suggest that, even in the entire dodecapeptide sequence corresponding to the loop of domain III of CaM, the calcium binding site is shifted toward the N-terminal hexapeptide segment. This interpretation is consistent with the results of crystallographic studies of CaM, which show that the calcium ions are located toward the amino terminal portion of the loop.     

Relations between ion shifting, ATP depletion and lactic acid formation in human red cells during moderate calcium loading using the ionophore A 23187.

Keeping constant cellular magnesium an A 23 187 mediated moderate calcium loading of human red cells causes isoosmotic cell shrinkage, potassium efflux, slight decrease of cellular pH, ATP depletion connected with an increase of AMP, ADP and Pi and enhanced lactic acid formation. The calcium loading and accompanying effects can be abolished by EGTA or by extracellular magnesium, the latter kept more than two orders of magnitude above that of calcium which was 30 micrometer. Inhibition of the (Mg2+ + Ca2+)-dependent ATPase by ruthenium red or lanthanum decreases the calcium stimulated lactic acid formation after a lag phase. However, the ATP depletion proceeds faster and is much more pronounced under these conditions. (Mg+2 + Na+ +K+)-dependent ATPase, hexokinase, phosphofructokinase and cell shrinkage are ruled out, too, as mediators of the ATP depletion. This suggests that an unknown ATP consuming reaction, apparently not being related to the calcium pump, causes the calcium induced ATP depletion.     

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.     

Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction.

Calcium ions are required for fusion of a wide variety of artificial and biological membranes. To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1. Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy. Syncytia did not form in solutions without calcium ions. Addition of calcium ions partially restored the formation of syncytia. EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] blocked syncytium formation in culture media containing calcium ions. Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions. Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM. Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+. Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+. We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.     

Energy transduction in Escherichia coli. The role of the Mg2+ATPase.

Inverted membrane vesicles from strain 7, a wild type Escherichia coli K12 strain, actively transport calcium with energy supplied either by respiration or by ATP. These vesicles also have energy-linked quenching of quinacrine fluorescence. Membranes of strain 7, depleted of Mg2+ATPase by EDTA treatment, lack both activities. Membrane vesicles from strain NR70, a mutant lacking the Mg2+ATPase, show neither calcium transport nor energy-linked fluorescence quenching. Neither EDTA treatment nor genetic loss of the Mg2+atpase causes a reduction in respiration. Purified Mg2+ATPase from strain 7 can bind to EDTA-treated membrane vesicles from either strain 7 or NR70. This binding restored both calcium transport and fluorescence quenching, driven either by respiration or by ATP. Dicyclohexylcarbodiimide treatment mimics the effect of the Mg2+ATPase in the case of respiration-driven reactions. Treatment with EDTA, while not essential for the binding of the Mg2+ATPase to membrane vesicles of NR70, produced better restoration of both activities. The rate of restoration of fluorescence quenching showed a time lag which may indicate that binding of the Mg2+ATPase is a relatively slow process. Antiserum prepared against the Mg2+ATPase inhibited the quenching of quinacrine fluorescence when driven by ATP but not when driven by respiration. Addition of antiserum prior to addition of Mg2+ATPase prevented the restoration of fluorescence quenching, whether driven by respiration or ATP. These results clearly show that MG2+ATPase has an important role not only in catalyzing ATP synthesis and hydrolysis but also in maintaining the energized membrane state.     

Modification of radiosensitivity of chlorpromazine with biological metal ions in Thiobacillus ferrooxidans

Effect of chlorpromazine with biological metal ions, viz. calcium, magnesium, zink and copper was studied on T. ferrooxidans cell system. Chlorpromazine, calcium and magnesium alone could produce radioprotection. Maximum radioprotection was exhibited by chlorpromazine at lower concentration while copper and zink offered radiosensitization. However, combination of chlorpromazine with all biological metal ions exhibited radiosensitization. Dose modifying factor by chlorpromazine at lower concentration (0.025 mM) was 0.754 while in combination with Ca2+, Mg2+, Cu2+ and Zn2+ was 1.08, 1.25, 1.37 and 1.389 respectively. The possible interaction between chlorpromazine and biological metal ions is discussed at cellular membrane level.     

Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.     

Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

Spectrophotometric determination of photoreceptor cGMP-gated channel Mg2(+)-fluxes using dichlorophosphonazo III.

We have characterised the spectroscopic properties of the metallochromic dye dichlorophosphonazo III and describe its use for the determination of changes of Mg2+ concentration in the micromolar range. Using a previously described reconstitution procedure, we incorporated the cGMP-gated channel from bovine rod photoreceptors into magnesium-containing liposomes and used the dye to monitor cGMP-activated Mg2(+)-efflux. The Km and cooperativity of the cGMP-dependence were identical regardless of whether Mg2+ or Ca2+ was the transported ion, however, the vmax for Ca2+ was more than 2-fold higher than that for Mg2+. We thereby determined a channel selectivity (Ca2+:Mg2+) of 1.0:0.44 in the presence of symmetrical (30 mM) K+. We also describe conditions where Mg2+ or Ca2+ effluxes can be selectively monitored in the presence of each other. This allowed the demonstration that magnesium ions can flow through the cGMP-gated channel even in the presence of an identically directed calcium gradient. Together these results indicate that magnesium ions may enter the photoreceptor rod outer segment cytosol through the cGMP-gated channel under dark conditions, thereby alluding to the existence of an as yet unknown Mg2(+)-extrusion mechanism, distinct from the Na+/Ca2(+)-exchanger, in these cells.     

Fluorescence proper of phospholipase A2 and orientotoxin from the venom of the giant hornet in the presence of ionic modulators of their membrane activity

The own fluorescence of phospholipase A2 and orientotoxin at interaction of these components from Oriental hornet venom with calcium, strontium, lantan, magnesium ions has been investigated. Ca2+, La3+, Sr2+--activators of phospholipase activity--increase the quantum output of fluorescence. In the presence of Ca2+ the temperature of semitransitional process of thermoinactivation of investigated polypeptides increases by 2 degrees C.     

Acute effect of hydrochlorothiazide on renal calcium and magnesium handling in postmenopausal women

A single 50 mg dose of hydrochlorothiazide (HCTZ) decreases the urinary excretion of calcium (U(Ca)V), clearance (C(Ca)) and fractional excretion (FE(Ca)) of calcium. This is accompanied by an increase of total calcium and ionized calcium (Ca2+) concentrations in the serum. On the other hand, HCTZ increases fractional excretion of magnesium (FE(Mg)) and decreases serum Mg2+ concentrations. Moreover, HCTZ decreases markedly clearance of phosphate (C(Pi)) and fractional excretion of phosphate (FE(Pi)) and increases serum phosphate (Pi) concentrations in healthy postmenopausal women. It is concluded that intrinsic renal cellular control promptly uncouples calcium and magnesium tubular reabsorption even without K+ depletion.     

Energy transduction in Escherichia coli. The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures.

1. The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium. 2. Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake. 3. The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide. 4. Chaotrope-treated membranes were found to lack Mg2+-ATPase activity. Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions. 5. Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents. 6. Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate. 7. It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase.     

The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Mg2+-induced changes of lipid order and conformation of (Na+ + K+)-ATPase

The effect of magnesium on the phospholipid order parameter and not the conformation of purified pig kidney outer medulla (Na+ + K+)-ATPase was investigated by fluorescence techniques. Measurements with a fluorescent probe TMA-DPH and its sensitized fluorescence with tryptophan residues as donors revealed that magnesium increased the order of the membrane phospholipids both in the lipid annulus and in the bulk phase. Changes in the lipid order induced by Mg2+ can be closely referred to the protein arrangement followed by the steady-state anisotropy of FITC-labeled (Na+ + K+)-ATPase.