The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

The Initiation of Spike Potential in Barnacle Muscle Fibers under Low Intracellular Ca++

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca⁺⁺-binding agents (K₂SO₄ and K salt of EDTA solution, K₃ citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca⁺⁺]_out. The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca⁺⁺ in the external medium, the spike potential is restored by Ba⁺⁺ and Sr⁺⁺ but not by Mg⁺⁺. The overshoot of the spike potential increases with increasing [Ba⁺⁺]_out or [Sr⁺⁺]_out. The Ca influx through the membrane of the fiber treated with K₂SO₄ and EDTA was examined with Ca⁴⁵. The influx was 14 pmol per sec. per cm² for the resting membrane and 35 to 85 pmol per cm² for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca⁺⁺ (Ba⁺⁺ or Sr⁺⁺).     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

Effect of lipid on the access of ATP and calcium to the delipidated Ca++-ATPase of intestinal brush border membrane.

The delipidated Ca⁺⁺-ATPase prepared from intestinal brush border membranes showed a higher activity of Ca⁺⁺-independent ATPase, a lower Km value for ATP and a higher Km value for Ca⁺⁺ than its original membrane Ca⁺⁺-ATPase. The addition of phosphatidylcholine re-activated the delipidated Ca⁺⁺-ATPase to approximately 89 % of its original membrane Ca⁺⁺-ATPase activity but did not restore the affinity for Ca⁺⁺. This phospholipid raised the Km value for ATP but had little effect on the Km value for Ca⁺⁺. Palmitic acid elevated the Km value for Ca⁺⁺ but did not change the Km value for ATP. Kinetic analyses of these data suggest that the hydrocarbon chain of phosphatidylcholine is an important rate-limiting factor for the access of Ca⁺⁺ to the enzyme and the polar head groups of phosphorylcholine and ester bond may be the factor for the access of ATP.     

Ba++ stimulates accumulation of cyclic AMP in rat pancreatic islets.

In pancreatic islets prelabelled with (³H) adenine, Ba⁺⁺ augmented (³H) cyclic AMP in 1–10 min incubations. 3-isobutyl-l-methylxanthine markedly enhanced and prolonged the Ba⁺⁺-induced nucleotide as well as the insulin response. In the presence of the methyl xanthine 1.6 mM Ba⁺⁺ was a maximally and 0.4 mM a submaximally effective concentration both for the stimulation of (³H) cyclic AMP and insulin. A 5-fold excess of Ca⁺⁺ partly inhibited the Ba⁺⁺-induced nucleotide and — more profoundly — the insulin response. Increasing Mg⁺⁺ from 2 to 10 mM was also inhibitory. Stimulation by Ba⁺⁺ was observed in the absence as well as in the presence of D-glucose. It is concluded that the insulinotropic action of Ba⁺⁺ is at least partly mediated by cyclic AMP.     

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig

Summary Ca⁺⁺-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca⁺⁺-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca⁺⁺ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca⁺⁺-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg⁺⁺ to the standard incubation medium, and 2) substituting Ca⁺⁺ with Mg⁺⁺ at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca⁺⁺ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany.     

Inhibition of Mg ++ ATPase activity of actin-activated Acanthamoeba myosin by muscle troponin-tropomyosin: implications for the mechanism of control of amoeba motility and muscle contraction

Troponin-tropomyosin is known to inhibit the Mg⁺⁺ATPase activity of muscle actomyosin in the absence, but not in the presence, of Ca⁺⁺. In contrast, we have now found that muscle troponin-tropomyosin inhibits the Mg⁺⁺ATPase activity of muscle actin-activated $\text{Acanthamoeba}$ myosin both in the presence and the absence of Ca⁺⁺. Addition of purified tropomyosin and troponins-I, C and T demonstrated that it is troponin-T that acts differently in the two systems which differ only in the source of the myosin. These data suggest that myosin, as well as actin, plays a role in the troponin-tropomyosin control of muscle contraction and make it unlikely that control proteins identical to troponin-tropomyosin function in this amoeba.     

A comparative study of the role of mitochondria and the sarcoplasmic reticulum in the uptake and release of Ca++ by the rat diaphragm

The respective importance of mitochondria and of sarcoplasmic reticulum in the uptake and maintenance of Ca⁺⁺ by the isolated rat diaphragm has been compared. Diaphragms were incubated at 30° in conditions optimal for Ca⁺⁺ uptake either by isolated mitochondria or by sarcoplasmic reticulum: more Ca⁺⁺ was taken up from the “mitochondrial” medium. For maximal uptake, Pi and Mg⁺⁺ were necessary; substitution of NaCl and KC1 with sucrose had no effect on the uptake. The uptake was markedly inhibited by uncouplers of oxidative phosphorylation, by respiratory inhibitors, and by lowering the temperature of the incubation medium to 0°; it was not affected by oligomycin, aurovertin, DCCD, nor by inhibitors of Ca⁺⁺ transport in the isolated sarcoplasmic reticulum (ergotamine, ergobasinine, caffeine). The lack of effect of caffeine was not due to lack of penetration into the muscle. Permeability barriers for ergotamine and ergobasinine could not be excluded. The maintenance of Ca⁺⁺ by the diaphragm was optimal in a medium contaming Pi and Mg⁺⁺. Uncoupling agents and respiratory inhibitors accelerated the rate and extent of release of Ca⁺⁺ by the diaphragm. Lowering the temperature of the incubation medium to 0°, or addition of oligomycin, aurovertin, DCCD, had no effect on the release. The release of Ca⁺⁺ was also unaffected by ergotamine, ergobasinine, caffeine. The results suggest a role for mitochondria in the uptake and maintenance of Ca⁺⁺ by the isolated diaphragm.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

Summary The histo- and cytochemical localization of Ca⁺⁺-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca⁺⁺-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca⁺⁺ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg⁺⁺ in different concentrations were substituted for Ca⁺⁺ in the incubation medium the reaction was always reduced. Both Ca⁺⁺ and Mg⁺⁺ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity is discussed in relation to the regulation of the extracellular Ca⁺⁺ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

THE AFFINITY OF ONION CELL WALLS FOR CALCIUM IONS

Cell walls prepared from onion bulbs were found to exhibit an affinity for Ca⁺⁺. The adsorption of this ion was enhanced by the action of pectin methylesterase. It was confirmed that Ca⁺⁺ reacts with two COO“ groups and the corresponding affinity constant, K, was found to be: log K = 4.25. The action of pectin methylesterase had no effect upon K. The cell walls, as prepared, had 25 % of the total COO⁻ groups occupied by Ca⁺⁺, 14 % by Mg⁺⁺, and 39 % by H⁺. Treatment with acidified ethanol removed all of the metallic cations. K⁺ and Mg⁺⁺ could displace Ca⁺⁺ from the cell walls. At concentrations from 10⁻³ to 3 times 10⁻³ m it required from 4.9 to 13.2 moles of Mg⁺⁺ to displace one mole of Ca⁺⁺. For K⁺ it required 80 moles to displace 1 mole of Ca⁺⁺ at K⁺ concentrations from 0.65 × 10⁻² to 1.6 × 10⁻² M.     

Molecular Dynamics of the Neuronal EF-Hand Ca2+-Sensor Caldendrin

Caldendrin, L- and S-CaBP1 are CaM-like Ca²⁺-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca²⁺-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca²⁺ and Mg²⁺ with a global K_d in the low µM range. Interestingly, the Mg²⁺-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg²⁺-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca²⁺-binding to Mg²⁺-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca²⁺-binding, high Mg²⁺- and low Ca²⁺-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca²⁺ of Mg²⁺-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca²⁺-sensors.     

Effect of mersalyl on mitochondrial Mg++ flux

The mercurial mersalyl has little effect either on rapid Mg⁺⁺ binding by isolated rat liver mitochondria or on the total Mg⁺⁺ content of these organelles measured after 0.75 min of incubation at 20°C. The data do not support the previous suggestion that the increased permeability to K⁺ of mitochondria treated with mersalyl results from release of endogenous Mg⁺⁺. An increased pH-dependence of unidirectional Mg⁺⁺ flux into respiring rat liver mitochondria is suggested to arise indirectly from inhibition by mersalyl of pH shifts associated with exchanges of endogenous phosphate. In addition, mersalyl appears to have a stimulatory effect on Mg⁺⁺ influx. Mersalyl also increases the average rate of unidirectional efflux of endogenous Mg⁺⁺. The stimulatory effects of mersalyl on Mg⁺⁺ flux are similar to, although quantitatively less than, the previously reported effects of mersalyl on mitochondrial K⁺ flux.     

Effect of chemical modification of cytoplasmic activator protein on human red cell membrane Ca++ + Mg+ ATPase.

A highly purified cytoplasmic activator protein of human red cell membrane Ca⁺⁺ + Mg⁺⁺ ATPase was prepared by two step purification scheme utilizing Diethylaminoethyl cellulose (DE-52) and sephadex (G-100) column chromatography. This purified protein can elicit a maximum activation of membrane Ca⁺⁺ + Mg⁺⁺ ATPase at low calcium concentrations. The stimulatory effect of this protein can be rendered totally ineffective by chemical modification with N-bromosuccinimide. The results suggest a possible role of methionine oxidation in the regulation of the Ca⁺⁺ + Mg⁺⁺ ATPase activator activity.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.