Revised model of calcium and magnesium binding to the bacterial cell wall

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca²⁺, Region I has a K_A = (1.0 ± 0.2) × 10⁶ M^?1 and Region II has a K_A = (0.075 ± 0.058) × 10⁶ M^?1. For Mg²⁺, K_A1 = (1.5 ± 0.1) × 10⁶ and K_A2 = (0.17 ± 0.10) × 10⁶. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η₂, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca²⁺ and Mg²⁺ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.     

Structures of cell wall teichoic acids of <Emphasis Type="Italic">Brevibacterium iodinum</Emphasis> VKM Ac-2106<Superscript>T</Superscript>

Structures of two cell wall teichoic acids of Brevibacterium iodinum VKM Ac-2106^T were studied. The structure of mannitol teichoic acid described earlier was mainly confirmed. This polymer is 1,6-poly(mannitol phosphate) bearing -D-glucopyranosyl residues at the C-2 of mannitol and pyruvic acid residues at the C-4 and C-5. The absolute configurations of D-mannitol and S-pyruvic acid were found. The following distinctions from the earlier described structure were found: unsubstituted 1,6-poly(mannitol phosphate) residues and residues substituted only by -D-glucopyranosyl at the C-2 of mannitol but unsubstituted by pyruvic acid are present in the chain. The structure of glycerol teichoic acid present in the cell wall as a minor component (7%) is also described. This acid is identified as 1,3-poly(glycerol phosphate) substituted at the C-2 of glycerol by 2-acetamido-2-deoxy--D-galactopyranosyl residues bearing R-pyruvic acid residues at the C-4 and C-6 of galactose. This polymer is for the first time described in the cell wall of Gram-positive bacteria.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1659–1666.Original Russian Text Copyright © 2004 by Potekhina, Evtushenko, Senchenkova, Shashkov, Naumova.     

The function of teichoic acids in cation control in bacterial membranes

1. The effects of teichoic acids on the Mg(2+)-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg(2+), showing maximum activity at 10-15mm-Mg(2+). 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg(2+). The membrane appears to interact preferentially with the constant concentration of Mg(2+) that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg(2+). 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg(2+) in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg(2+); this preparation contained Mg(2+) bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg(2+). Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg(2+) requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.     

A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis

Bacterial cell wall homeostasis is an intricately coordinated process that ensures that envelope integrity is maintained during cell growth and division, but can also adequately respond to growth‐limiting conditions such as phosphate starvation. In Bacillus subtilis, biosynthesis of the two major cell wall components, peptidoglycan and anionic polymers, is controlled by a pair of paralogous two‐component systems, WalRK and PhoPR respectively. Favorable growth conditions allow for a fast rate of cell wall biosynthesis (WalRK‐ON) and the incorporation of the phosphate‐containing anionic polymer teichoic acids (PhoPR‐OFF). In contrast, growth‐restricted cells under phosphate‐limiting conditions reduce the incorporation of peptidoglycan building blocks (WalRK‐OFF) and switch from the phosphate‐containing teichoic acids to the phosphate‐free anionic polymer teichuronic acid (PhoPR‐ON). Botella et al. (2014) deepen our knowledge on the PhoPR system by identifying one signal that is perceived by its histidine kinase PhoR. In fast‐growing cells, intracellular intermediates of teichoic acid biosynthesis are sensed by the cytoplasmic Per‐Arnt‐Sim domain as an indicator of favorable conditions, thereby inhibiting the autokinase activity of PhoR and keeping the system inactive. Depletion of teichoic acid building blocks under phosphate‐limiting conditions relieves this inhibition, activates PhoPR‐dependent signal transduction and hence the switch to teichuronic acid biosynthesis.     

Inhibition by Chloramphenicol of Glucose Transfer in Teichoic Acid Biosynthesis

CHLORAMPHENICOL is widely accepted as a highly effective inhibitor of protein synthesis in bacteria, both in whole cells and at the subcellular level. Although some of the details of its mechanism of action are still unsettled, it has been shown to bind selectively to the 50S ribosomal subunit¹ and to inhibit peptide formation possibly by preventing binding between the ribosome and raRNA². It is well established that the bacteriostatic action of the antibiotic results from inhibition of protein synthesis and its use as a tool in the study of cellular biochemistry is frequently based on the view that its action is highly specific. In fact, there are reports of its inhibitory action on other cell processes³, but these are either relatively unimportant or the effects are only shown at concentrations of antibiotic much higher than those required for inhibition of protein synthesis. Anraku and Landman⁴ have reported that chloramphenicol inhibits a late stage in the reversion of protoplasts of Bacillus subtilis to the osmotically stable bacillary form and this is accompanied by inhibition of synthesis of a phosphorylated wall polymer believed to be a teichoic acid. It was suggested that inhibition of synthesis of wall polymers, including the teichoic acid, was an indirect effect arising from inhibition of synthesis of the appropriate enzyme proteins. We now report that chloramphenicol powerfully inhibits the biosynthesis of a wall teichoic acid in a cell-free system of fragmented cytoplasmic membrane from B. licheniformis ATCC 9945 (B. subtilis NCIB 8062); this occurs through direct action on the teichoic acid synthesizing system and is unrelated to protein synthesis. Although this may provide an alternative explanation of the effect observed by Anraku and Landman, a more detailed study of their system would be required.     

The biosynthesis and functionality of the cell-wall of lactic acid bacteria

The cell wall of lactic acid bacteria has the typical Gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attribut es of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.     

Studies on the activation of isocitrate dehydrogenase kinase/phosphatase (AceK) by Mn<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg²⁺. We have studied the interactions of Mn²⁺and Mg²⁺ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn²⁺ also activates the enzyme activities. However, Mn²⁺ and Mg²⁺ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?10⁵ and 0.98?×?10⁵ M^?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn²⁺ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn²⁺ and Mg²⁺.     

Cell wall structure and function in lactic acid bacteria

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.     

The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids

We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a σ^A-dependent promoter and situated in the 308° chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymerexport systems of Gram-negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N-moiety contains the ATP-binding motif and shares extensive homology with a number of ATP-binding proteins, particularly with those associated with the transport of capsular polysaccharides and O-antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5′ -moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.     

Calmodulin can induce and control damped oscillations in plasma membrane Ca<Superscript>2+</Superscript>-ATPase activity: A kinetic model

Plasma membrane Ca²⁺-ATPase is the pump that extrudes calcium ions from cells using ATP hydrolysis to maintain low Ca²⁺ concentrations in the cell. Calmodulin stimulates Ca²⁺-ATPase by binding to the autoinhibitory enzyme domain, which allows the access of cytoplasmic ATP and Ca²⁺ to the catalytic and transport sites. Our kinetic model predicts damped oscillations of the enzyme activity and interprets the known nonmonotonic kinetic behavior of the enzyme in the presence of calmodulin. For parameters close to experimental data, the kinetic model explains the dependence of the frequency and damping factor of the oscillatory enzyme activity on the calmodulin concentration. The calculated pre-steady-state curves fit well to known experimental data. Kinetic analysis allows us to assign Ca²⁺-ATPase to hysteretic enzymes exhibiting activity oscillations in open systems.     

Effect of Mg<Superscript>2+</Superscript> versus Ca<Superscript>2+</Superscript> on the behavior of Annexin A5 in a membrane-bound state

Annexin A5 (AnxA5) binds to negatively charged phospholipid membranes in a Ca²⁺ dependent manner. Several studies already demonstrate that Mg²⁺ ions cannot induce the binding. In this paper, quartz crystal microbalance with dissipation monitoring (QCM-D), Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) and molecular dynamics (MD) were performed to elucidate the high specificity of Ca²⁺ versus Mg²⁺ on AnxA5 binding to membrane models. In the presence of Ca²⁺, AnxA5 showed a strong interaction with lipids, the protein is adsorbed mainly in α-helix under the DMPS monolayer, with an orientation of the α-helices axes slightly tilted with respect to the normal of the phospholipid monolayer as revealed by PMIRRAS. The Ca²⁺ ions interact strongly with the phosphate group of the phospholipid monolayer. In the presence of Mg²⁺, instead of Ca²⁺, no interaction of AnxA5 with lipids was detected. Molecular dynamics simulations allow us to explain the high specificity of calcium. Ca²⁺ ions are well exposed and surrounded by labile water molecules at the surface of the protein, which then favour their binding to the phosphate group of the membrane, explaining their specificity. To the contrary, Mg²⁺ ions are embedded in the protein structure, with a smaller number of water molecules strongly bound. We conclude that the embedded Mg²⁺ ions inside the AnxA5 structure are not able to link the protein to the phosphate group of the phospholipids for this reason.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Isolation of the Teichoic Acid of Bacillus Subtilis 168 by Affinity Chromatography

A column of insoluble concanavalin A was prepared by coupling the protein to cyanogen bromide-activated Sepharose. When autolysates of Bacillus subtilis 168 cell walls were passed over the column, the alpha glucosylated teichoic acid component of the cell wall was retained. The teichoic acid could be eluted with dilute alpha-methylglucopyranose. The teichoic acid prepared by affinity chromatography from cell wall autolysates had a higher sedimentation rate than teichoic acids obtained by conventional methods.

Several authors have shown that concanavalin A (con A) forms complexes with alpha-glucosylated teichoic acids^1–3. Doyle and Birdsell¹ found that the teichoic acid of Bacillus subtilis 168 (trp C2) would precipitate with con A at neutral pH in dilute buffer. The formation of a precipitate was inhibited by sugars which bind to the active site of con A. This observation suggested that it should be possible to purify the teichoic acid by affinity chromatography using insoluble con A as the affinity probe. Lloyd⁴ and Donnelly and Goldstein⁵ have successfully employed insoluble con A to purify polysaccharides and glycoproteins. In this communication, we describe conditions for the rapid purification of the alpha-glucosylated teichoic acid of B. subtilis 168. The teichoic acid prepared by this procedure appears to be less degraded than teichoic acids obtained by conventional methods.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Late-stage polyribitol phosphate wall teichoic acid biosynthesis in Staphylococcus aureus

Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.     

Free Mg<Superscript>2+</Superscript> concentration in the calf muscle of glycogen phosphorylase and phosphofructokinase deficiency patients assessed in different metabolic conditions by <Superscript>31</Superscript>P MRS

Background

The increase in cytosolic free Mg²⁺ occurring during exercise and initial recovery in human skeletal muscle is matched by a decrease in cytosolic pH as shown by in vivo phosphorus magnetic resonance spectroscopy (³¹P MRS). To investigate in vivo to what extent the homeostasis of intracellular free Mg²⁺ is linked to pH in human skeletal muscle, we studied patients with metabolic myopathies due to different disorders of glycogen metabolism that share a lack of intracellular acidification during muscle exercise.

Methods

We assessed by ³¹P MRS the cytosolic pH and free magnesium concentration ([Mg²⁺]) in calf muscle during exercise and post-exercise recovery in two patients with McArdle's disease with muscle glycogen phosphorylase deficiency (McArdle), and two brothers both affected by Tarui's disease with muscle phosphofructokinase deficiency (PFK).

Results

All patients displayed a lack of intracellular acidosis during muscle exercise. At rest only one PFK patient showed a [Mg²⁺] higher than the value found in control subjects. During exercise and recovery the McArdle patients did not show any significant change in free [Mg²⁺], while both PFK patients showed decreased free [Mg²⁺] and a remarkable accumulation of phosphomonoesters (PME). During initial recovery both McArdle patients showed a small increase in free [Mg²⁺] while in PFK patients the pattern of free [Mg²⁺] was related to the rate of PME recovery.

Conclusion

i) homeostasis of free [Mg²⁺] in human skeletal muscle is strongly linked to pH as shown by patients' [Mg²⁺] pattern during exercise;ii) the pattern of [Mg²⁺] during exercise and post-exercise recovery in both PFK patients suggests that [Mg²⁺] is influenced by the accumulation of the phosphorylated monosaccharide intermediates of glycogenolysis, as shown by the increased PME peak signal.iii) ³¹P MRS is a suitable tool for the in vivo assessment of free cytosolic [Mg²⁺] in human skeletal muscle in different metabolic conditions;

     

Effects of multivalent cations on cell wall-associated Acid phosphatase activity

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu²⁺, Mg²⁺, Zn²⁺, and Mn²⁺; unaffected by Ba²⁺, Cd²⁺, and Pb²⁺; and inhibited by Al³⁺. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg²⁺. On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al³⁺ and Hg²⁺ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg²⁺. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca²⁺ significantly reduced the effects of Hg²⁺ or Al³⁺, but not Mg²⁺, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg²⁺ or Al³⁺ which caused a Ca²⁺-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.     

The mechanism of neuroprotection by positive modulation of Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels of cerebellar neurons in primary culture

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.     

Increase of free Mg<Superscript>2+</Superscript>in the skeletal muscle of chronic fatigue syndrome patients

In a previous study we evaluated muscle blood flow and muscle metabolism in patients diagnosed with chronic fatigue syndrome (CFS). To better understand muscle metabolism in CFS, we re-evaluated our data to calculate free Magnesium levels in skeletal muscle. Magnesium is an essential cofactor in a number of cell processes. A total of 20 CFS patients and 11 controls were evaluated. Phosphorus magnetic resonance spectroscopy from the medial gastrocnemius muscle was used to calculate free Mg²⁺ from the concentrations and chemical shifts of Pi, PCr, and beta ATP peaks. CFS patients had higher magnesium levels in their muscles relative to controls (0.47 + 0.07 vs 0.36 + 0.06 mM, P < 0.01), although there was no difference in the rate of phosphocreatine recovery in these subjects, as reported earlier. This finding was not associated with abnormal oxidative metabolism as measured by the rate of recovery of phosphocreatine after exercise. In summary, calculation of free Mg²⁺ levels from previous data showed CFS patients had higher resting free Mg²⁺ levels compared to sedentary controls.     

Effects of fatty acids on calcium-stimulated adenosine triphosphatase in rat submandibular gland microsomes.

Effects of fatty acids on Ca²⁺-ATPase and Mg²⁺-ATPase in the microsomal fraction of rat submandibular gland have been investigated. Saturated fatty acids had almost no effect, but unsaturated fatty acids inhibited both ATPases. Modes of inhibition by linoleic acid were as follows: competitive for calcium and ATP with Ca²⁺-ATPase; non-competitive for magnesium and ATP with Mg²⁺-ATPase