Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

Optimum conditions for the activation of the alternative complement pathway of a cyprinid fish (Tinca tinca L.). Seasonal variations in the titres

The present work describes the existence of a haemolytic activity in the serum of tench, Tinca tinca, against rabbit red blood cells (RRBC) which was identified as belonging to the alternative complement pathway from the following findings: haemolytic activity disappeared when the serum was heated to 45°C for 20 min; 10 mM EDTA, which chelates Ca²⁺ and Mg²⁺, induced a complete loss of haemolysis; Mg²⁺, but not Ca²⁺, was required for the activity, and the use of sheep red blood cells (SRBC), which have a high content of sialic acid, resulted in the serum activity falling to a very low degree of haemolysis. The ACH50 value (units ml^-1 serum) was defined as the reciprocal of the serum dilution necessary to lyse 50% of 4 × 10⁷ RRBC in a buffered medium of normal ionic strength (μ=0·15) containing 10 mM EGTA and optimum concentrations of Mg²⁺. The optimum conditions for the ACH50 assay were: pH 7·2-7-7; reaction temperature, 15°C; concentration of Mg²⁺, 5 mM; and reaction time, 90 min. Under these conditions, the values of ACH50 in spring, summer, autumn and winter for male tench were 69±13, 91±22, 90±36 and 137±41, and for female tench 100±11, 108±13, 82±12 and 145±17. The highest serum activity was found in the winter, suggesting the importance of this pathway during cold periods when the specific immune response is depressed in ectothermic vertebrates.     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

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     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

Interaction of Anions and Cations in Regulating Energy Distribution between the Two Photosystems

Cations such as Mg²⁺ regulate spillover of absorbed excitation energy mainly in favour of photosystem (PS) 2. Effect of low concentration (<10 mM) of the monovalent cation Na⁺ on chlorophyll (Chl) a fluorescence was completely overridden by divalent cation Mg²⁺ (5 mM). Based on Chl a fluorescence yield and 77 K emission measurements, we revealed the role and effectiveness of anions (Cl^-, SO₄ ^2-, PO₄ ^3-) in lowering the Mg²⁺-induced PS2 fluorescence. The higher the valency of the anion, the lesser was the expression of Mg²⁺ effect. Anions may thus overcome Mg²⁺ effects up to certain extent in a valency dependent manner, thereby diverting more energy to PS1 even in the presence of MgCl₂. They may do so by reversing Mg²⁺-induced changes.     

The Ribosomal E Site at Low Mg2+: Coordinate Inactivation of Ribosomal Functions at Mg2+ Concentrations Below 10 mM and its Prevention by Polyamines

Abstract

Under standard conditions (Mg²⁺/150 mM NH₄ ⁺) ribosomes can quantitatively participate in tRNA binding at Mg²⁺ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P,E or A sites decrease in a parallel manner when the Mg²⁺ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg²⁺, and is completely blocked at 6—8 mM.

The severe inactivation seen at 6 mM Mg²⁺ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg²⁺ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus,J.Biol. Chem. 261, 9133 (1986)) are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites).

The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg²⁺/NH₄ ⁺/polyamine- system seems to be the best compromise at present between these two requirements.     

Salt-Induced L-Forms of Staphylococcus aureus

When growing cultures of a salt-sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 m NaCl and 0.5% bovine serum albumin, typical colonies of L-form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L-form induction.     

Magnesium deprivation inhibits a MEK–ERK cascade and cell proliferation in renal epithelial Madin-Darby canine kidney cells

AimsLoss of magnesium (Mg²⁺) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg²⁺ has not been clarified in detail. We examined the effect of extracellular Mg²⁺ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg²⁺-containing or Mg²⁺-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg²⁺ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg²⁺]_i. Re-addition of Mg²⁺ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg²⁺. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg²⁺. These results indicate that the MEK–ERK cascade is regulated by [Mg²⁺]_i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg²⁺, but was inhibited by Mg²⁺ deprivation. Mg²⁺ deprivation did not increase the number of dead cells.SignificanceMg²⁺ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells.     

Transmembrane Mg2+ Currents and Intracellular Free Mg2+ Concentration in Paramecium tetraurelia

The properties of Mg²⁺ conductances in Paramecium tetraurelia were investigated under two-electrode voltage clamp. When bathed in physiological Mg²⁺ concentrations (0.5 mm), depolarizing steps from rest elicited a prominent Mg²⁺-specific current (I _Mg) that has been noted previously. The dependence of this current on extracellular Mg²⁺ approximated that of Mg²⁺-induced backward swimming, demonstrating that I _Mg contributes to normal membrane excitation and behavior in this ciliate. Closer analysis revealed that the Mg²⁺ current deactivated biphasically. While this might suggest the involvement of two Mg²⁺-specific pathways, both tail-current components were affected similarly by current-specific mutations and they had similar ion selectivities, suggesting a common pathway. In contrast, a Mg²⁺ current activated upon hyperpolarization could be separated into three components. The first, I _Mg, had similar properties to the current activated upon depolarization. The second was a nonspecific divalent cation current (I _NS) that was revealed following suppression of I _Mg by eccentric mutation. The final current was relatively minor and was revealed following suppression of I _Mg and I _NS by obstinate A gene mutation. Reversal-potential analyses suggested that I _Mg and I _NS define two intracellular compartments that contain, respectively, low (0.4 mm) and high (8 mm) concentrations of Mg²⁺. Measurement of intracellular free Mg²⁺ using the fluorescent dye, Mag-fura-2, suggested that bulk [Mg²⁺] _i rests at around 0.4 mm in Paramecium. Received: 12 January 1998/Revised: 16 March 1998     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

Summary Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca²⁺ currents found in this cell. The peak of the low threshold current (channel I) occurred at –20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca²⁺ and Mg²⁺ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca²⁺ or Mg²⁺ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×10⁶ m ^–1 sec^–1 for Mg²⁺ and 4.5×10⁸ m ^–1 sec^–1 for Ca²⁺. The Ca²⁺ block was somewhat relieved at negative potentials, whereas the Mg²⁺ block was not, suggesting that Ca²⁺, but not Mg²⁺, can exit from the binding site toward the cell interior.     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

Effects of Ca and Mg on Growth and Calcification of the Coccolithophorid Pleurochrysis haptonemofera: Ca Requirement for Cell Division in Coccolith-Bearing Cells and for Normal Coccolith Formation with Acidic Polysaccharides

The effects of Ca²⁺ and Mg²⁺ on cellular growth and calcification in Pleurochrysis haptonemofera were investigated. In the presence of a normal concentration of Mg²⁺, coccolith-bearing cells (C-cells) required more than 0.5 mM Ca²⁺ for growth, while naked cells could grow even with 0.5 mM Ca²⁺. The calcification rate of C-cells, which was determined using decalcified cells, was significantly repressed with less than or equal to 0.5 mM Ca²⁺. Although the calcification rate did not change so much with 5–30 mM Ca²⁺, it decreased with higher concentrations of Ca²⁺, as well as C-cell-specific growth repression. Under these conditions, Ca²⁺ affected the rate of coccolith formation, but neither the coccolith morphology nor total amounts and ratios of divalent cations and acidic polysaccharides (Ph-PS-1, -2, and -3) were included in coccoliths. These findings suggest that sufficient calcification is required for the division of C-cells. Under low Ca²⁺ and high Mg²⁺ conditions, coccoliths with an abnormal morphology, having immature shield elements, were synthesized. Composition analysis of the coccoliths revealed high Mg/Ca and low Ph-PS-2/(Ph-PS-1 and -3) ratios, as compared with those under low Ca²⁺ and normal Mg²⁺ conditions, suggesting that the abnormal morphology is due to a change in the crystal type and/or acidic polysaccharide composition.     

The binding of Mg++ ions to polyadenylate, polyuridylate, and their complexes

The binding of Mg ⁺⁺ to polyadenylate (poly A), Polyuridylate(poly U), and their complexes, poly (A + U) and poly (A + 2U), was studied by means of a technique in which the dye eriochrome black T is used to measure the concentration of free Mg^?. The apparent binding constant K_X = [MgN]/[Mg⁺⁺][N], N = site for Mg⁺⁺ binding (the phosphate group of the nucleotide), was found to decrease rapidly as the extent of binding increased and, at low extents of binding, as the concentration of Na^? increased in poly A, poly (A + U), and poly (A + 2U), and somewhat less so in poly U. K_x is generally in the range 10⁴ > K_X > 10². The cause of these dependences is apparently, primarily, the displacement of Na⁺ by Mg⁺⁺ in poly U and poly (A + U) on the basis of the similarity of extents of displacement measured in this work and those measured potentiometrically. was calculated and was found to approach zero as the concentration of Na⁺ increased. In poly U, poly (A + U), and poly (A + 2U) at low ΔH′ v.H. > 0, about + 2 kcal/“mole.” In poly A, also at low salt, ΔH′ v.H. ≈ ?4 kcal/“mol” for the initial binding of Mg⁺⁺, and increases to +2 kcal/“mol” at saturation. This enthalpic variation probably accounts for the anticooperativity in the binding of Mg⁺⁺ not ascribable to the displacement of Na⁺⁺.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll light-harvesting antenna.     

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.     

Role of magnesium in the (Ca2++Mg2+)-stimulated membrane ATPase of human red blood cells

Summary (Ca²⁺+Mg²⁺)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca²⁺ concentration and at two different but constant Mg²⁺ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value forK _m (for ATP) of 1–2m is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975.J. Membrane Biol. 22:313). Mg²⁺ increases both the maximal rate and the affinity for ATP, whereas Ca²⁺ increases the maximal rate without affectingK _m for ATP.As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1mm substrate concentration is several times faster than maximal rate of (Ca²⁺+Mg²⁺)-ATPase in red cell membranes.     

Novel function of eubacterial flagella: role in aggregation of a marine bacterium

Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN₃. Aggregates were not observed when Mg²⁺ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca²⁺, Mn²⁺, and Ba²⁺ could replace Mg²⁺. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs⁺ or Li⁺ instead of Mg²⁺. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.Abbreviation ASW artificial seawater