Acid-facilitated supramolecular assembly of G-quadruplexes in d(CGG)4

Molar [K+] induces aggregate formation in d(CGG)4, as evidenced by absorbance, circular dichroic (CD), and gel measurements. The kinetics of this transformation are extremely slow at pH 8 but are found to be greatly facilitated in acidic conditions. Kinetic profiles via absorbance or CD monitoring at single wavelength resemble those of autocatalytic reacting systems with characteristic induction periods. More than 0.8 M KCl is needed to observe the onset of aggregation at 20 degrees C and pH 5.4 within the time span of 1 day. Time-dependent CD spectral characteristics indicate the formation of parallel G-tetraplexes prior to the onset of aggregation. Despite the evidence of K(+)-induced parallel G-quadruplex and higher molecular weight complex formation, both d(TGG)4 and d(CGG)4T fail to exhibit the observed phenomenon, thus strongly implicating the crucial roles played by the terminal G and base protonation of cytosines. A plausible mechanism for the formation of a novel self-assembled structure is speculated. Aided by the C+.C base pair formation, parallel quadruplexes are initially formed and subsequently converted to quadruplexes with contiguous G-tetrads and looped-out cytosines due to high [K+]. These quadruplexes then vertically stack as well as horizontally expand via interquadruplex C+.C base pairing to result in dendrimer-type self-assembled super structures.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.     

Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin

The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.     

Primary angioplasty for acute myocardial infarction

The comparative stability of eight different triplexes constituted with 16-mer pyrimidine-modified oligodeoxynucleotides (wild-type ODN, PS-ODN, alpha-ODN, or alpha-PS-ODN) or oligoribonucleotides (wild-type ORN, alpha-ORN, 4'-thio-ORN, or 2'-O-MeORN) and a DNA hairpin, termed H36, was studied in five different buffers by UV melting curve analysis. The composition of buffers varied in pH (5.5 and 6.5), in salt concentration (100 mM and 1 M Na+), and in the presence or absence of divalent cation (0 or 3 mM Mg2+) or spermine (0 or 1 mM). At pH 5.5, the eight triplexes are formed with Tm values ranging from 24.7 degrees C to 50.9 degrees C (delta G298K between -8.1 and -16.8 kcal/mol). At pH 6.5, the triplexes are less stable, and thus 4'-thio-ORN and PS-ODN showed broad transitions that did not allow us to conclude triplex formation. An increase of salt concentration or the presence of spermine stabilizes the triplexes, whereas Mg2+ has a destabilizing effect (excepted for alpha-ORN). In general ORN:H36 and 2'-O-MeORN:H36 triplexes were the most stable. Finally, introduction of alpha-anomeric nucleosides led to an alpha-ORN analog that showed low binding with H36 and to alpha-ODN and alpha-PS-ODN analogs. Triplexes formed with alpha-ODN were slightly less stable than those formed with unmodified ODN. Surprisingly, introduction of phosphorothioate in an alpha analog led only to a low destabilization.     

Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme

Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.     

Regulation of intracellular calcium concentrations by calcium and magnesium in cardioplegic solutions protects rat neonatal myocytes from simulated ischemia

The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37 degrees C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+ concentration (0.1, 0.6, 1.2, or 2.4 mM), the Mg2+ concentration was either 0, 1.2, 8, or 16 mM. The increase in intracellular Ca2+ concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+ concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+ accumulation. However, in the 0.1 mM Ca2+ groups in which the Mg2+ concentration was greater than 8 mM, the intracellular Ca2+ concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+ and Mg2+ present in cardioplegic solutions control the intracellular Ca2+ concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution.     

Vasodilative response to hypoxia and simulated ischemia is mediated by ATP-sensitive K+ channels in guinea pig thoracic aorta

Local vasodilation in response to hypoxia or ischemia improves perfusion and O2 supply of the affected tissue. This local vasodilation thus constitutes the most important mechanism in the prevention of ischemic cell injury. The regulation of vascular tone has mainly been attributed to changes of cytoplasmatic Ca2+ ((Ca2+)i) concentrations in vascular smooth muscle cells. The mechanism underlying these changes has not, however, been elucidated so far. Using aortic strips of guinea pigs (transversally cut in spirals; normal Tyrode, in mM: NaCl 150, KCl 4.5, MgCl2 2, CaCl2 2.5, glucose 10; buffered with 10 mM HEPES at pH 7.4; equilibrated with 100% O2 at 31 degrees C) the authors could show that metabolic blockade (glucose replaced by 10 mM 2-deoxyglucose (DOG) led to a relaxation of the preparation. Thus, in four experiments, resting tension decreased from 0.75 g by 27% +/- 12% within two hours (% of maximal contractile force developed by each preparation when depolarized with 43 mM KCl and 101.5 mM NaCl). When the same experiment was carried out in the presence of 1 mM tolbutamide (a known blocker of ATP-dependent K+ channels) in vascular smooth muscle no such relaxation could be seen (n = 4). Furthermore, in the same type of preparation, similar results have been obtained upon hypoxic relaxation (100% O2 replaced by 100% N2), where 1 mM tolbutamide also prevented vasodilation. Thus, hypoxic/ischemic vasodilation in response to glycolytic inhibition (DOG) and hypoxia (N2) is based upon the opening of K+ ATP channels and hence can be prevented by sulfonylureas (the opening of K+ ATP channels would lead to hyperpolarization (increased K+ conductance, Goldmann equation), thus diminishing the open probability of voltage-gated Ca2+ channels with subsequent vasodilation). This inhibition by sulfonylureas of vasodilative response to ischemia may also constitute the so far unknown cause of the increased cardiovascular mortality seen under sulfonylurea treatment.     

Effect of the substituted benzaldehyde 12C79 on Cl--dependent K+ influx in human red blood cells

Ouabain- and bumetanide-resistant K+ influx, and haemoglobin (Hb) O2 saturation, were measured in HbA red cells over a range of oxygen tensions (PO2 values) in the presence and absence of 12C79 (5 mM), a substituted benzaldehyde which increases the O2 affinity of Hb. PO2 values for half-maximal O2 saturation declined from 29+/-2 mmHg (mean +/-SEM, n=3) in control cells to 7+/-1 mmHg with 12C79. In control cells, Cl--dependent K+ influx (indicative of KCl cotransport activity) was fully O2 dependent, i.e. inactive at low PO2 values. By contrast, in the presence of 12C79, KCl cotransport was largely resistant to inactivation at low PO2 values. Substantial cotransport activity was still present (>60% of that at high PO2 values) in N2, although O2 saturation was low (about 10%). In all cases, Cl--independent K+ influxes were low [<0.25 mmol (l cells h)-1] and unaffected by PO2 or 12C79. The significance of these results is discussed.     

Changed expression of 9-O-acetyl GD3 (CDw60) in benign and atypical proliferative lesions and carcinomas of the human breast

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (> or = 300 mM).     

Dual mechanisms of Mg2+ block of ryanodine receptor Ca2+ release channel from cardiac sarcoplasmic reticulum

We investigated the effects of cytosolic Mg2+ on ryanodine receptor Ca2+ release channel (RyR) of bovine cardiac sarcoplasmic reticulum incorporated into planar lipid bilayers recording single channel activities. Channels were activated by > or = 0.1 microM Ca2+ in the cis solution. At constant Ca2+, application of Mg2+ (0.1-1 mM) to cis side decreased channel activity in a concentration-dependent manner. A half maximal blocking concentration (Kd) was 35 microM and a complete block was obtained at 1 mM. In the presence of 1 mM free Mg2+ in cis solution, the relation between the channel open probability (Po) and concentration of free Ca2+ in cis solution ([Ca2+]cis) shifted to the right, indicating the competition of Mg2+ and Ca2+. Blocking effects of Mg2+ on RyR were antagonized by increasing [Ca2+]cis > or = 0.1 mM. In the presence of 1 m Mg2+ and 1 mM Ca2+ in cis solution, the channel conductance was markedly depressed to approximately 400 pS (n = 7) from 603 +/- 40 pS (mean +/- S.D., n = 22) in the absence of Mg2+, indicating the flickering block. These results show that Mg2+ causes a direct inhibition of RyR in cardiac SR and this inhibition may be mediated through two different mechanisms. A competition of Mg2+ and Ca2+ at a Ca2+ sensitive site on the RyR and a flickery block of the open channel by Mg2+.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

Magnesium enhances function of postischaemic human myocardial tissue

OBJECTIVES: The effect of Mg2+ on the developed force and concentrations of high energy phosphate metabolites in isolated human atrial trabeculae has been investigated. METHODS: Human atrial trabeculae, obtained from right atrial appendages of patients undergoing cardiac surgery requiring cardiopulmonary bypass, were dissected at room temperature in modified Krebs-Henseleit buffer containing 1.2 or 16 mM Mg2+, mounted on muscle stands, and rewarmed to 34 degrees C in the same buffer. After 30 minutes, their mechanical function was assessed. At the end of the protocol, trabeculae were fast frozen for measurement of concentrations of metabolites of high energy phosphates. RESULTS: Trabeculae collected and rewarmed in 16 mM Mg2+ Krebs-Henseleit buffer showed significantly higher mean developed force (0.59(SEM 0.10) g, p < 0.01) than those rewarmed in 1.2 mM Mg2+ Krebs-Henseleit buffer (0.32(0.03) g). Trabeculae that had a developed force > or = 0.8 g, a resting force < or = 0.7 g, and a cross sectional area < or = 1 mm2 ("functional" trabeculae) were selected for further comparison. New reverse phase high performance liquid chromatography techniques developed for the analysis of small samples (0.5-5 mg dry weight) were used to measure nucleotide, nucleoside, and creatine compounds. Total adenylate (ATP+ADP+AMP) concentrations in trabeculae revived in the presence of 16 mM Mg2+ (15.4(1.1) mumol.g-1 dry weight) were significantly higher (p < 0.01) than in those revived with 1.2 mM Mg2+ (11.8(1.0) mumol.g-1), but lower (p < 0.01) than in trabeculae fast frozen immediately after removal from the patient (22.6(1.0) mumol.g-1). There were no significant differences in NAD and total creatine (phosphocreatine+creatine) concentrations between the three groups. CONCLUSIONS: The presence of high Mg2+ during the rewarming of human atrial trabecular preparations maintains a significantly higher developed force and a significantly higher total adenylate pool than does collection and rewarming with normal concentrations of Mg2+.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on glucose isomerase from a Streptomyces species

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.     

Mg2+ inhibition of ATP-activated current in rat nodose ganglion neurons: evidence that Mg2+ decreases the agonist affinity of the receptor

The effect of Mg2+ on ATP-activated current in rat nodose ganglion neurons was investigated with the use of the whole cell patch-clamp technique. Mg2+ decreased the amplitude of ATP-activated current in a concentration-dependent manner over the concentration range of 0.25-8 mM, with a 50% inhibitory concentration value of 1.5 mM for current activated by 10 microM ATP. Mg2+ shifted the ATP concentration-response curve to the right in a parallel manner, increasing the 50% effective concentration value for ATP from 9.2 microM in the absence of added Mg2+ to 25 microM in the presence of 1 mM Mg2+. Mg2+ increased the deactivation rate of ATP-activated current without changing its activation rate. The observations are consistent with an action of Mg2+ to inhibit ATP-gated ion channel function by decreasing the affinity of the agonist binding site on these receptors.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.