Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

The (Na + K)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl2

Na⁺-dependent ADP/ATP exchange activity, of a (Na⁺ + K⁺)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of ¹⁴C into ATP during incubations with unlabeled ATP and [¹⁴C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl₂ concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg²⁺, Mg · ATP, and Mg · ADP present, with a range of MgCl₂, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg²⁺, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg²⁺, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg²⁺, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.     

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

Changes in action potentials and intracellular ionic homeostasis in a ventricular cell model related to a persistent sodium current in SCN5A mutations underlying LQT3

In LQT3 patients, SCN5A mutations induce ultraslow inactivation of a small fraction of the hNav1.5 current, i.e. persistent Na⁺ current (I_pNa). We explored the time course of effects of such a change on the intracellular ionic homeostasis in a model of guinea-pig cardiac ventricular cell [Pasek, M., Simurda, J., Orchard, C.H., Christé, G., 2007b. A model of the guinea-pig ventricular cardiomyocyte incorporating a transverse–axial tubular system. Prog. Biophys. Mol. Biol., this issue]. Sudden addition of I_pNa prevented action potential (AP) repolarization when its conductance (g_pNa) exceeded 0.12% of the maximal conductance of fast I_Na (g_Na). With g_pNa at 0.1% g_Na, the AP duration at 90% repolarization (APD₉₀) was initially lengthened to 2.6-fold that in control. Under regular stimulation at 1 Hz it shortened progressively to 1.37-fold control APD₉₀, and intracellular [Na⁺]_i increased by 6% with a time constant of 106 s. Further increasing g_pNa to 0.2% g_Na caused an immediate increase in APD₉₀ to 5.7-fold that in control, which decreased to 2.2-fold that in control in 30 s stimulation at 1 Hz. At this time diastolic [Na⁺]_i and [Ca²⁺]_i were, respectively, 34% and 52% higher than in control and spontaneous erratic SR Ca release occurred.

In the presence of I_pNa causing 46% lengthening of APD₉₀, the model cell displayed arrhythmogenic behaviour when external [K⁺] was lowered to 5 mM from an initial value at 5.4 mM. By contrast, when K⁺ currents I_Kr and I_Ks were lowered in the model cell to produce the same lengthening of APD₉₀, no proarrhythmic behaviour was observed, even when external [K⁺] was lowered to 2.5 mM.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Gill microsomal (Na+,K+)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na⁺,K⁺)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na⁺ binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K⁺. The binding of K⁺ ions to their high affinity sites displaces Na⁺ ions from their sites. The increase in Na⁺ ion concentrations increases heterotropic interactions with the K⁺ ions, with no changes in K_0.5 for K⁺ ion activation at the extracellular sites. Differently from mammalian (Na⁺,K⁺)-ATPases, that from C. danae exhibits additional NH₄⁺ ion binding sites that synergistically activate the enzyme at saturating concentrations of Na⁺ and K⁺ ions. NH₄⁺ binding is cooperative, and heterotropic NH₄⁺ ion interactions are insensitive to Na⁺ ions, but Na⁺ ions displace NH₄⁺ ions from their sites. NH₄⁺ ions also displace Na⁺ ions from their sites. Mg²⁺ ions modulate enzyme stimulation by NH₄⁺ ions, displacing NH₄⁺ ion from its sites. These interactions may modulate NH₄⁺ ion excretion and Na⁺ ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.     

The effect of hydroxylamine on microsomal ATPase

The (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase, but not the Mg²⁺-ATPase, is irreversibly inhibited when turtle bladder microsomes were incubated with hydroxylamine.

The Mg²⁺-dependent or the (Mg²⁺ + Na⁺)-dependent phosphorylation of ADP by the phospho-protein (the exchange reaction) is reversibly inhibited when the microsomes are incubated with hydroxylamine.

The Na⁺-induced increment of ³²P-labelling of microsomes previously incubated with [λ-³²P]ATP is completely eliminated by hydroxylamine, but the Mg²⁺-dependent ³²P-labelling of such microsomes is unaffected by hydroxylamine.

It is concluded that the phospho-enzyme formed during the Mg²⁺-dependent hydrolysis does not contribute to the Mg²⁺-dependent exchange reaction. Instead, the phosphoenzyme formed during the (Na⁺ + K⁺)-stimulated hydrolysis is apparently the only substance which phosphorylates ADP in the exchange reaction, even in the absence of Na⁺ and/or K⁺.

The hydroxylamine-sensitive nature of the sodium form of the phospho-enzyme in the (Na⁺ + K⁺)-stimulated ATPase sequence is consistent with the existence of an enzyme-acyl-phosphate bond of high internal energy with respect to that of ADP.

On the other hand, the hydroxylamine-resistant nature of the phospho-enzyme in the Mg²⁺-ATPase sequence suggests the existence of a non-acyl type of enzyme phosphate bond with low internal energy relative to that of ADP.     

磁化微咸水灌溉对欧美杨I-107离子稳态的影响

为探究微咸水磁化处理条件下植株的离子稳态特征,以欧美杨I-107一年生扦插苗为试材,于生长季节分别采用Hoagland营养液和4.0 g·L^-1 NaCl微咸水,经磁化处理后连续灌溉30 d.采用原子吸收分光光度法对叶片和根系中K⁺、Na⁺、Ca²⁺和Mg²⁺含量进行测定,分析离子平衡系数(K)和根-叶之间的离子选择性运输系数(S_Xi,Na).结果表明: 与非盐分胁迫处理相比,盐分胁迫处理根系和叶片中Na⁺和Ca²⁺含量及S_K,Na、S_Mg,Na升高,K⁺和Mg²⁺含量、K⁺/Na⁺及S_Ca,Na降低.与非磁化微咸水灌溉处理相比,磁化微咸水灌溉处理的根系和叶片中Na⁺含量降低、K⁺含量及K⁺/Na⁺提高;根系和叶片中Ca²⁺含量降低、Mg²⁺含量提高;磁化微咸水灌溉处理中K提高,且叶片中K值显著高于根系;S_K,Na和S_Mg,Na较非磁化微咸水灌溉提高,S_Ca,Na较其降低.磁化微咸水灌溉中根系和叶片Na⁺积累量减少,K⁺、Ca²⁺和Mg²⁺含量增加,且维持了较高水平的K⁺/Na⁺,这有利于植株整株水平生理代谢的调控.因此,盐分胁迫下磁化作用可通过调节离子的选择性吸收和运输来维持植株体内的离子平衡.     

H2O2 and Src-dependent transactivation of the EGF receptor mediates the stimulatory effect of leptin on renal ERK and Na+, K+-ATPase

We examined the mechanism through which leptin increases Na⁺, K⁺-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na⁺, K⁺-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na⁺, K⁺-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H₂O₂ excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na⁺, K⁺-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H₂O₂ and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H₂O₂ increased Src phosphorylation at Tyr⁴¹⁸. We conclude that leptin-induced stimulation of renal Na⁺, K⁺-ATPase involves H₂O₂ generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK.     

Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes

The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn²⁺. The EPR-detected Mn²⁺ competition studies give an order of apparent affinity of Mn²⁺ Co²⁺ Zn²⁺>Cd²⁺Mg²⁺. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn²⁺>Co²⁺>Cd²⁺>Mg²⁺. The maximum rate is observed for Mn²⁺ under these conditions and may be related to the high affinity, low pK_a and low ΔH_hyd of this ion. Substitution of phosphorothioates 5′ to each of the nine adenosines in the enzyme strand yields a change in the Mn²⁺ binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn²⁺ bind in two types of classes: ‘type 1’ (n=1±0.3, K_d=1.1±1 μM) and weaker ‘type 2’ (n=7.7±0.3, K_d=125±27 μM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd²⁺>Zn²⁺>Co²⁺, Mg²⁺ with the number of Mn²⁺ displaced and apparent affinity of the thiophilic Cd²⁺ most affected by the phosphorothioate substitutions.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Metal ion-coordinating properties of imidazole and derivatives in aqueous solution: interrelation between complex stability and ligand basicity

The stability constants of the 1:1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Cd²⁺ (M²⁺) and the simple, sterically unhindered imidazole-type ligands, imidazole, 1-methylimidazole, 5-chloro-1-methylimidazole, N-(2,3,5,6-tetrafluorophenyl)imidazole or 4′-(imidazol-1-yl)acetophenone (L), were determined by potentiometric pH titrations in aqueous solution (25°C; I = 0.5 M, NaNO₃). The construction of log K_ML^M versus pK_HL^H plots results in straight lines; the equations for the least-squares lines are calculated and listed. These data allow calculation of the expected stability constant for a complex of any imidazole-type ligand, provided its pK_HL^H value (in the pK_a range 4–8) is known. For the stabilities of Fe²⁺ complexes with imidazole-type ligands an estimation procedure is provided. It is shown further that the complex formation between 1-methylbenzimidazole (MBI) and Mn²⁺, Ni²⁺, Cu²⁺ or Zn²⁺ is s sterically hindered, i.e. the data points for these M(MBI)²⁺ complexes do not fall on the straight lines defined by the imidazole-type ligands.