Effect of magnesium ion (Mg2+) and magnesium adenosinetriphosphate ion (MgATP2-) on pigeon kidney pyruvate carboxylase

Kinetic methods have been used to determine whether Mg²⁺ and MgATP^2? play an important role in regulating pigeon kidney pyruvate carboxylase (pyruvate: CO₂ ligase (ADP), EC 6.4.1.1.). Mg²⁺ not only forms a complex with ATP^4? (MgATP^2?) but is also required for the enzyme activation (and probably for the binding of MgATP^2? to this enzyme). Contrary to the results of other investigators, the MgATP^2? complex was not found to activate pigeon kidney pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP^2? complex. Excess Mg²⁺ induces allosteric stimulation of the enzymatic activity at different concentrations of MgATP^2?. With different Mg²⁺ concentrations, changes also occured in the apparent Km^? and Vmax-values. Without excess of Mg²⁺ only about 2 % of the total enzymic activity available could be demonstrated in the presence of MgATP^2?. It is concluded that Mg²⁺ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg²⁺ may bind either to a specific effector site, at the active site, or at the binding site for MgATP^2? which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate.     

Cytosolic Phosphofructokinase from Spinach Leaves : II. Affinity for Mg and Nucleoside Phosphates

Cytosolic ATP-phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was inhibited by submillimolar concentrations of free Mg²⁺. The free Mg²⁺ concentration required for 50% inhibition of PFK activity was 0.22 millimolar. Inhibition by free Mg²⁺ was independent of the MgATP²⁻ concentration. Inorganic phosphate (Pi) reduces the inhibition of PFK activity by Mg²⁺. Free ATP (ATP⁴⁻) also inhibits PFK activity. For free ATP the inhibition of PFK activity was dependent on the MgATP²⁻ concentration. Fifty percent inhibition of PFK activity requires 1.2 and 3.7 millimolar free ATP at 0.1 and 0.5 millimolar MgATP²⁻, respectively. It was proposed that free ATP competes for the MgATP²⁻ binding site, whereas free Mg²⁺ does not. Pi diminished the inhibitory effect of free ATP on PFK activity. Free ATP and Pi had substantial effects on the MgATP²⁻ requirement of cytosolic PFK. For half-maximum saturation of PFK activity 3 and 76 micromolar MgATP²⁻ was required at 0.007 and 0.8 millimolar free ATP in the absence of Pi. At 5 and 25 millimolar Pi, half-maximum saturation was achieved at 9 and 14 micromolar MgATP²⁻. PFK activity was inhibited by Ca²⁺. The inhibition by Ca²⁺ depends upon the total Mg²⁺ concentration. Fifty percent inhibition of PFK activity required 22 and 32 micromolar Ca²⁺ at 0.1 and 0.2 millimolar Mg²⁺, respectively. At physiological concentrations of about 0.5 millimolar free Mg²⁺, Ca²⁺ would have little effect on cytosolic PFK activity from spinach leaves. PFK is not absolutely specific for the nucleoside 5′-triphosphate substrate. Besides MgATP²⁻, MgUTP²⁻, MgCTP²⁻, and MgGTP²⁻ could be used as a substrate. All four free nucleotides inhibit PFK activity. The physiological consequences of the regulatory properties of cytosolic PFK from spinach leaves will be discussed. A model will be introduced, in an attempt to describe the complex interaction of PFK with substrates and the effectors Mg²⁺ and Pi.     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Kinetic characterization of barley root plasma membrane-bound Ca2+- and Mg2+-dependent adenosine triphosphatase activities

A plasma membrane-rich microsome fraction isolated from barley (Hordeum vulgare L. cv. Conquest) roots contained considerable divalent cation-dependent ATPase activity when assayed at 16°C. The maximal divalent cation-stimulation of the apparent basal ATPase activity varied as Ca²⁺ > Mg²⁺ > Mn²⁺= Zn²⁺ > Co²⁺ > Ni²⁺, with all other divalent cations tested being inhibitory. Double reciprocal plots of the Ca²⁺- and Mg²⁺-dependent ATPase velocities as a function of substance concentration were nonlinear, suggesting the presence of multiple catalytic sites. Both MgATP^2- and CaATP^2- served as the true substrates and apparently bind to the same catalytic sites. Free ATP and Ca²⁺ could inhibitit the Ca²⁺- and Mg²⁺-dependent ATPase. Increasing free Mg²⁺ levels enhanced the affinity of the Mg²⁺-dependent ATPase for MgATP^2-, while slightly inhibiting the V_max values. Other divalent cation-nucleoside triphosphate complexes produced maximal enzyme velocities equal to or greater than those generated by CaATP^2- and MgATP^2-. However, the ATPase had significantly higher affinities for CaATP^2- and MgATP^2-, than for the alternative substrates. The high and low affinity components of the Ca²⁺- and Mg²⁺-dependent ATPase exhibited optimal V_max values at pH 5 and 6, respectively. Analysis of the pH-dependence of the enzyme K_m values indicated enzyme-substrate binding with charge neutralization at neutral and alkaline pH's. Nonlinear double reciprocal plots were obtained at all assay temperatures. However, the complexity of the enzyme kinetics became less apparent at the higher assay temperatures. The kinetics of the barley root divalent cation-dependent ATPase activities are discussed in terms of the kinetics of ATPases from other plants and the methods used to obtain them, and compared to the kinetics of ion transport ATPases from animal membranes.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Purification and Characterization of a 4-Hydroxybenzoate Decarboxylase from <Emphasis Type="Italic">Chlamydophila pneumoniae</Emphasis> AR39

Chlamydophila pneumoniae AR39 is an obligate intracellular pathogen that causes human acute and chronic respiratory tract diseases. One protein from C. pneumoniae AR39 was assigned as 4-hydroxybenzoate decarboxylase (HBDC). Assays done with the purified oxygen-sensitive protein showed that the optimum pH and temperature were 7.5 and 30°C, respectively. The Km and Vmax obtained for 4-hydroxybenzoate were approximately 0.21 mM and 11.9 nM min⁻¹ mg⁻¹, respectively. During the period of 4-hydroxybenzoate decarboxylation, overall activity of the thermal-sensitive protein was 5.06 nM min⁻¹ mg⁻¹ protein. The 4-hydroxybenzoate decarboxylation was promoted by Mg²⁺, Fe²⁺, Mn²⁺, and Ca²⁺ but not by Cu²⁺ or Zn²⁺. The enzyme also slowly catalyzed the reverse reaction, which was phenol carboxylation.     

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⁺.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Characterization of trehalose synthase from <Emphasis Type="Italic">Corynebacterium nitrilophilus</Emphasis> NRC

Trehalose synthase (TSII) from Corynebacterium nitrilophilus NRC was successively purified by ammonium sulphate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-100 columns. The specific activity of the trehalose synthase was increased ~200-fold, from 0.14 U mg⁻¹ protein to 28.3 U mg⁻¹ protein. TSII was found to be a monomeric protein with a molecular weight of 67–69 kDa. Characterization of the enzyme exhibited optimum pH and temperature were 7.5 and 35°C, respectively. The purified enzyme was stable from pH 6.6 to 7.8 and able to prolong its thermal stability up to 35°C. The enzyme activity was inhibited strongly by Zn²⁺, Hg²⁺ and Cu²⁺ and moderately by Ba²⁺, Fe²⁺, Pb²⁺ and Ni²⁺. Other metal ions Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺ and EDTA had almost no effect.     

Spatio-Temporal Distributions of Metal Ions and Taxol of Taxus cuspidata Cells Immobilized on Polyurethane Foam

Distinct spatio-temporal variations of metal ions and Taxol production were observed for Taxus cuspidata cells immobilized on polyurethane foam. The Taxol content in the inner foam layer reached 215 μg g⁻¹ at day 30, which was 40-fold higher than that in the outer foam layer, and the Ca²⁺ and Mg²⁺ contents were 5.3 and 3.7 times higher, while the K⁺ content was 5.5 times lower. Thus higher intracellular Ca²⁺ and Mg²⁺ contents and lower intracellular K⁺ content may favor the Taxol biosynthesis in immobilized Taxus cuspidata.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: III. Modulation of ATPase Activity by Reaction Substrates and Products

Two distinct membrane fractions containing H⁺-ATPase activity were prepared from red beet. One fraction contained a H⁺-ATPase activity that was inhibited by NO₃⁻ while the other contained a H⁺-ATPase inhibited by vanadate. We have previously proposed that these H⁺-ATPases are associated with tonoplast (NO₃⁻-sensitive) and plasma membrane (vanadate-sensitive), respectively. Both ATPase were examined to determine to what extent their activity was influenced by variations in the concentration of ATPase substrates and products. The substrate for both ATPase was MgATP²⁻, and Mg²⁺ concentrations in excess of ATP had only a slight inhibitory effect on either ATPase. Both ATPases were inhibited by free ATP (i.e. ATP concentrations in excess of Mg²⁺) and ADP but not by AMP. The plasma membrane ATPase was more sensitive than the tonoplast ATPase to free ATP and the tonoplast ATPase was more sensitive than the plasma membrane ATPase to ADP.

Inhibition of both ATPases by free ATP was complex. Inhibition of the plasma membrane ATPase by ADP was competitive whereas the tonoplast ATPase demonstrated a sigmoidal dependence on MgATP²⁻ in the presence of ADP. Inorganic phosphate moderately inhibited both ATPases in a noncompetitive manner.

Calcium inhibited the plasma membrane but not the tonoplast ATPase, apparently by a direct interaction with the ATPase rather than by disrupting the MgATP²⁻ complex.

The sensitivity of both ATPases to ADP suggests that under conditions of restricted energy supply H⁺-ATPase activity may be reduced by increases in ADP levels rather than by decreases in ATP levels per se. The sensitivity of both ATPases to ADP and free ATP suggests that modulation of cytoplasmic Mg²⁺ could modulate ATPase activity at both the tonoplast and plasma membrane.

     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Purification and Characterization of an Esterase from <Emphasis Type="Italic">Acinetobacter lwoffii</Emphasis> I6C-1

EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K _m ) and V _max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min⁻¹ mg of protein-1, respectively. The enzyme was strongly inhibited by Hg²⁻, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Co²⁺, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP). Received: 20 August 2001 / Accepted: 20 September 2001     

Effects of divalent metal cations on lovastatin biosynthesis from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> in chemically defined medium

Effects of six divalent metal cations: Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺, Cu²⁺and Mn²⁺ on fungal cell growth and lovastatin biosynthesis were investigated by submerged cultivation of Aspergillus terreus in a modified chemically defined medium. The influences of different initial concentrations of the above six metal cations were also examined at 1, 2, and 5 mM, respectively. Cu²⁺ apparently inhibited the cell growth, but had no influence on biosynthesis of lovastatin. All of Fe²⁺, Ca²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ promoted the cell growth and lovastatin biosynthesis in different extents. The highest biomass of 13.8 ± 0.5 g l⁻¹ and specific lovastatin titres of 49.2 ± 1.4 mg gDCW⁻¹ were obtained at the level of 2 and 5 mM in the presence of Zn²⁺, respectively. The values were improved double and 14.4-fold. Excess Zn²⁺ inhibited the cell growth, but enhanced lovastatin biosynthesis with an increment of 17.6 mg l⁻¹ per mM. The interactions of all metal cations slightly inhibited the lovastatin production comparing with the existence of Zn²⁺, Fe²⁺ and Mg²⁺ solely, yet remarkably improved the cell growth. These results suggest that the divalent metal ions Zn²⁺ or Fe²⁺ influence the production by regulating the action of key enzymes such as LovD or LovF in lovastatin biosynthesis.     

Investigation on the micro-mechanisms of Al3+ interfering the reactivities of aspartic acid and its biological processes with Mg2+

Density functional theory (DFT) has been applied to study the micro-mechanisms of Al³⁺ interfering the reactivities of aspartic acid (H₂asp) and its biological processes with Mg²⁺. All the 46 stable conformers of Hasp^- and 3 of asp²⁻ have been determined at the B3LYP/6-311++G** level, showing that the 7 most stable conformers of Hasp⁻ all present a very strong and linear O–H···O H-bond between carboxyl and carboxylic acid groups with the bond energy high up to 162 kJ mol⁻¹. The reaction thermodynamics and micro-mechanism between Al³⁺ and Hasp⁻ (or asp²⁻) in aqueous phase have been investigated by the combined application of supramolecular model and polarizable continuum IEFPCM solvent model, firstly revealing Al³⁺ interfering in the biological processes of aspartic acid. The substitution thermodynamics and mechanisms of Mg²⁺ by Al³⁺ in the biological processes between the species of aspartic acid and Mg²⁺ in aqueous phase were probed, revealing the facile displacement of Mg²⁺ by Al³⁺. These results may provide a reasonable mechanism of Al³⁺ biological toxicity at the microscopic level.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Interaction of MgATP2− with DNA: Assessment of metal binding sites and DNA conformations by spectroscopic and thermal denaturation studies

Spectroscopic (IR, UV, CD and fluorescence) and thermal denaturation studies of native calf thymus DNA, DNAMgATP²⁻ and DNAMg²⁺ have been carried out in aqueous KBr medium (introduced by the present authors as a very effective solvent for DNA). The IR data recorded for the systems indicate that MgATP²⁻ binds to the N₇ and C₆O of the guanine residue of DNA forming a five-membered chelate ring. The data also suggest that despite binding to the guanine bases, Mg²⁺ binds more strongly to the phosphate moiety of DNA. Solution CD spectra of DNA, DNAMgATP²⁻ and DNAMg²⁺ indicate that in each case DNA exists in the B conformation. Thin-film CD studies reveal that irrespective of the relative humidity conditions, pure DNA as well as that after interaction with Mg²⁺ show a structural transition B → C, conformationally, although belonging to the B family. A similar study shows that DNA on interaction with MgATP²⁻ assumes a more packed conformation (B)_n giving rise to a ψ⁻ spectrum. Steady-state as well as dynamic fluorimetric studies clearly indicate that MgATP²⁻ does not intercalate between CGGC base pairs. The thermal denaturation studies support the IR data with respect to the metal binding sites and the mode of binding in both cases.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.