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.     

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.     

The effect of divalent metal ions on the activity of Mg++ depleted ribulose-1,5-bisphosphate oxygenase

Ribulose-1,5-bisphosphate carboxylase-oxygenase is deactivated by removal of Mg⁺⁺. The enzyme activities can be restored to a different extent by the addition of various divalent ions in the presence of CO₂. Incubation with Mg⁺⁺ and CO₂ restores both enzyme activities, whereas, the treatment of the enzyme with the transition metal ions (Mn⁺⁺, Co⁺⁺, and Ni⁺⁺) and CO₂ fully reactivates the oxygenase: however, the carboxylase activity remains low. In experiments where CO₂-free conditions were conscientiously maintained, no reactivation of RuBP oxygenase was observed, although Mn⁺⁺ ions were present. Other divalent cations such as Ca⁺⁺ and Zn⁺⁺, restore neither the carboxylase nor the oxygenase reaction. Furthermore, the addition of Mn⁺⁺ to the Mg⁺⁺ and CO₂ preactivated enzyme significantly inhibited carboxylase reactions, but increased the oxygenase reaction.Abbreviation RuBP ribulose-1,5-bisphosphate. The enyme unit for RuBP carboxylase is defined as mol CO₂ fixed·min^-1 and for the RuBP oxygenase as mol O₂ consumed · min^-1     

Coordination of Mn++ ions at contact sites between tRNA and aminoacyl-tRNA synthetase.

By means of PMR and ESR study the shielding of Mn⁺⁺ ions by aminoacyl-tRNA synthetase has been detected in the aminoacyl-tRNA synthetase - tRNA complex at pH 7.5. At pH 6 this effect was not observed. We propose that ions interact with certain aminoacyl-tRNA synthetase groups protonated when passing to slightly acid pH. The role of Mn⁺⁺ and Mg⁺⁺ ions in the formation of a functionally active complex tRNA-aminoacyl-tRNA synthetase is discussed.     

Modulation of single cardiac Na+ channels by cytosolic Mg++ ions

Elementary Na⁺ currents were recorded in inside-out patches excised from cultured neonatal rat heart myocytes in order to study the influence of cytosolic Mg⁺⁺ and other bivalent cations present at the cytoplasmic membrane surface on cardiac Na⁺ channel gating. Exposing the cytoplasmic membrane surface to a Mg⁺⁺-free environment shortened the open state of cardiac Na⁺ channels significantly. _open declined to 62±2% of the value obtained at 5 mmol/l Mg_i ⁺⁺. Other channel properties including the tendency to reopen and the elementary current size either changed insignificantly within a 10% range or remained completely unchanged. An almost identical change of _open can be caused by switching from a Mn⁺⁺ (5 mmol/l) containing internal solution to a Mn⁺⁺-free internal solution. But _open failed to significantly respond to a variation in internal Ni⁺⁺ from 5 mmol/l to 0 mmol/l. The same response to internal Mg⁺⁺ withdrawal was obtained with (–)-DPI-modified, non-inactivating Na⁺ channels, indicating that the exit rate from the open state remains as sensitive to cytosolic Mg⁺⁺ variations as in normal Na⁺ channels with operating inactivation. Offprint requests to: M. Kohlhardt     

The effect of Mg ++ on the conformation of the Ca ++ -binding component of troponin

Mg⁺⁺ like Ca⁺⁺ induces a conformational change in the Ca⁺⁺-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 80 % of the change in optical rotation induced by Ca⁺⁺. The apparent binding constant of Mg⁺⁺ to the Ca⁺⁺-binding component is 5 × 10³ M⁻¹, much smaller than that of Ca⁺⁺. Circular dichroism measurements show that these changes are simple helix-coil transitions. Unlike the Ca⁺⁺-induced conformational change, the Mg⁺⁺-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.     

Cation activation of desoxyribonuclease

The activation of desoxyribonuclease on desoxyribonucleate, known to occur with Mg⁺⁺ and Mn⁺⁺, has been shown to occur equally well with Co⁺⁺, to nearly the same extent with Fe⁺⁺, and to a lesser extent with Ca⁺⁺, Ba⁺⁺, Sr⁺⁺, Ni⁺⁺, Cd⁺⁺, and Zn⁺⁺. The conditions under which the optimal activation is revealed vary among these ions. Thus, Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ may show marked activation under conditions in which Fe⁺⁺ is nearly ineffective. Since too high a concentration of an ion may be as ineffective as too little, concentration-activation curves were determined for each ion. Per micromole of nucleic acid phosphorus, the optimal effective amount of each ion in micromoles is as follows: Mg⁺⁺ 3, Mn⁺⁺ 3, Co⁺⁺ 3, Fe⁺⁺ 0.3, Ni⁺⁺ 0.3, Ba⁺⁺ 1.7, Ca⁺⁺ 3, Sr⁺⁺ 3, Zn⁺⁺ 0.3, and Cd⁺⁺ 0.3.The optimum pH for the activation with Mg⁺⁺, Co⁺⁺, and Ca⁺⁺ is about 6.5, that with Fe⁺⁺ is at 5.7, while Mn⁺⁺ shows two optima at pH 6.8 and 8.0.Experiments conducted in Pyrex and in quartz vessels showed the same results, and indicated that there was no activation of desoxy-ribonuclease in the absence of added salts.     

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.     

New ATP incorporating activities from rat liver mitochondria: the attachment of a portion of ATP to a pronase-sensitive receptor

Studies using a Brij 58 detergent extract of rat liver mitochondria reveal that these organelles can catalyze the time-dependent incorporation of a portion of [³H]ATP into an acid-insoluble product. The activities studied using 8 mM Mn⁺⁺ or 15 mM Mg⁺⁺ are stimulated by dithiothreitol and by CTP, GTP or UTP, while that studied using 2 mM Mg⁺⁺ is not. The incorporated tritium remains bound after incubation in the presence of excess unlabeled ATP and chromatography on Sephadex G-25. The labeled product is insensitive to ribonuclease A and snake venom phosphodiesterase, but is sensitive to pronase. The attached portion of the ATP molecule released upon treatment of the product has been tentatively identified as adenosine for the activities studied using 2 mM Mg⁺⁺ or 8 mM Mn⁺⁺ and as AMP (80%) and adenosine (20%) for the reaction studied using 15 mM Mg⁺⁺.     

Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

Interaction of metallic cations with DNA VI. Specific binding of Mg++ and Mn++

The binding of Mg²⁺ and Mn²⁺ by DNA by a divalent cation specific electrode and by ultracentrifugation. Both techniques give similar results for the stoichiometry of the reaction. An oscillating densiemete allowed us to detect small changes of volume accompanying the binding. The reaction was also followed by circular dichroism measurements. Interpretation of the results is only possible if one assumes an electrostate site-binding of Mg²⁺ to phosphate group, and a chelation Mn²⁺ between the phosphate group and the N₇ of the guanine. Physical modifications accompanying these two types of binding are discused and compared to the role of these cations in some biological systems involving DNA.     

23Na NMR investigation of the interaction between DNA and divalent metallic cations

The aim of this study is to follow the thermodynamic behaviour of Na⁺ ions, acting as natural counterions of DNA, in the presence of divalent metal ions, by using the²³Na NMR technique. With the help of the²³Na entropy of fluctuations concept introduced by Lenk, we propose the following decreasing sequence: Mg⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, and Cu⁺⁺, for the magnitude of divalent metal ions interactions with DNA phosphate sites.     

Ion and pH gradients across the transport membrane of mitochondria following Mn ++ uptake in the presence of acetate

The electron paramagnetic resonance (EPR) spectrum of Mn⁺⁺ loaded mitochondria is affected by the presence of the permeant anion acetate (Ac^?) in the medium. The hyperfine sextet, shown earlier to have spectral characteristics like those expected of osmotically active Mn⁺⁺ in the matrix space, grows in intensity with increasing [Ac^?]. From estimates of mitochondrial water, the free internal [Mn⁺⁺] can be calculated. The gradient of free [Mn⁺⁺] across the inner mitochondrial membrane is believed to be at least 500:1 under conditions of high [Ac^?]. Since Mn⁺⁺ solubility is limited by [OH^?], it is possible to place an upper limit on the pH in the matrix space. The variation of free internal [Mn⁺⁺], as measured by EPR, with external pH indicates that the [H⁺] gradient is 1–1.5 pH units in the absence of permeant anions and considerably less in the presence of 100 mM acetate.     

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.     

Kinetic studies of adenylyl cyclase of fat cell membranes

Summary The kinetic behavior of the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation on sucrose gradients was studied. Under most of the conditions explored, with either Mn⁺⁺ or Mg⁺⁺ as the divalent cation in the assay mixtures, the time courses of the reaction were not linear. In the absence of modifiers (i.e., basal activity) or in the presence of insulin, the rate tended to decrease with time; on the other hand, with fluoride or GMP-P(NH)P the curves were concave upwards. To simplify analysis of the results, two kinetic components were defined: an initial component corresponding to the transient rate measured between zero time and 1.5 min of assay and a final component corresponding to the transient rate determined between 3 and 5 min.Over the entire range of Mn⁺⁺ concentration explored (0.5 to 6.0mm), the basal initial rates were slightly higher than the final ones. With Mg⁺⁺ in the range between 1.5 and 2.5mm, the final rates were fourfold lower than the initial ones. Higher or lower Mg⁺⁺ concentrations gave velocity ratios equivalent to those observed with Mn⁺⁺.Insulin clearly decreased the final rates at Mn⁺⁺ concentrations up to 2.5mm. With higher concentrations the effects were completely reversed. The effects of insulin on initial rates measured with Mn⁺⁺, or the initial or final rates measured with Mg⁺⁺, were less evident.Stimulation of adenylyl cyclase activity by fluoride was most pronounced on the final rates. In addition, this stimulation was higher with Mg⁺⁺ than with Mn⁺⁺.Isoproterenol stimulation of adenylyl cyclase was negligible in the presence of Mn⁺⁺ (0.5 to 6.0mm). With Mg⁺⁺ (0.5 to 6.0mm), stimulation was more evident on the final rates. *** DIRECT SUPPORT *** A0130063 00002     

Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions

Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn⁺⁺ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg⁺⁺-stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg⁺⁺ and Mn⁺⁺ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca⁺⁺-free Ringer-fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg⁺⁺-stimulated activity by 24% and Mg⁺⁺?+ Mn⁺⁺-stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.     

Phosphoglycerate mutase in developing forespores of Bacillus megaterium may be regulated by the intrasporal level of free manganous ion.

When young intact forespores of Bacillus megaterium were incubated with either Mn⁺⁺ or the ionophore X-537A, the pool of 3-phosphoglyceric acid (3-PGA) was stable. However, incubation of forespores with Mn⁺⁺ plus the ionophore X-537A resulted in rapid and complete utilization of the 3-PGA. This effect was not seen with Ca⁺⁺ or Mg⁺⁺, and was also not observed with older forespores or fresh dormant spores. Since the phosphoglycerate mutase of $\text{B.}\text{megaterium}$ has an absolute and specific requirement for Mn⁺⁺, it is possible that phosphoglycerate mutase in developing forespores may be inactive because of a low intrasporal level of free Mn⁺⁺.     

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.