Increase in the stoichiometry of the functioning active sites of horse liver aldehyde dehydrogenase in the presence of magnesium ions

The V of horse liver aldehyde dehydrogenase is enhanced twofold in the presence of 0.5 mm Mg²⁺ ions when assayed in the dehydrogenase reaction. The mechanism of this activation appears to be related to the fact the enzyme changes from functioning with half-of-the-sites reactivity to functioning with all-of-the-sites reactivity. That is, the presteady-state burst magnitude increases from 2 mol NADH formed per mole of tetrameric enzyme to 4 mol formed per mole (K. Takahashi and H. Weiner, J. Biol. Chem., 1980, 255, 8206–8209). Whether this twofold enhancement correlates, in fact, to a change from half-of-the-sites to all-of-the-sites reactivity of the enzyme by Mg²⁺ ions was investigated by determining the Stoichiometry of coenzyme binding by fluorescence quenching and enhancement methods in the absence and presence of the metal ions. The biphasic Scatchard plots for NAD binding to the enzyme were similar in the absence and presence of Mg²⁺ ions, while that of NADH binding was monophasic (-Mg²⁺) and biphasic (+Mg²⁺). In the presence of p-methoxyacetophenone, a competitive inhibitor for substrate, the stoichiometric titration of coenzyme binding to the ternary complexes (enzyme-NAD(H)-inhibitor) revealed that only 2 mol of NAD or NADH bind in the absence of Mg²⁺ ions but 4 bind per mole of tetrameric enzyme in the presence of added metal. The fluorescence intensity of NAD's fluorescent derivative, 1,N⁶-ethenoadenine dinucleotide, bound to the enzyme was also doubled by the addition of Mg²⁺ ions.The combined binding data show that the stoichiometry of coenzyme binding to aldehyde dehydrogenase in the ternary complex increases from 2 to 4 mol binding per mole of tetrameric enzyme with the addition of Mg²⁺ ions. This increase in stoichiometry corresponds to the observed changes of burst magnitude obtained from the presteady-state and V in the steady-state kinetics assays. From both results of the kinetics and stoichiometry, we show that horse liver aldehyde dehydrogenase exhibits half-of-the-sites reactivity when in the tetrameric state in the absence of Mg²⁺ ions, and all-of-the-sites reactivity in the dimeric state in the presence of the metal.     

The effects of magnesium and calcium ions on phosphatase activities at alkaline pH in the molar region of newborn mice

Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca²⁺ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg²⁺/Ca²⁺ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg²⁺ ions were added. No ATPase activity at all could be recorded in media containing Mg²⁺ but no Ca²⁺ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca²⁺ or Mg²⁺ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells.     

Kinetic studies of bovine liver carbamoyl phosphate synthetase

A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO₃⁻, ATPMg and NH₄⁺. A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.     

35Cl nuclear magnetic resonance study of zinc and phosphate binding of E. coli alkaline phosphatase

³⁵Cl^? quadrupole relaxation was measured in the presence of metal-free alkaline phosphatase and in the presence of Zn²⁺-alkaline phosphatase. The relaxation data show that for an enzyme containing the minimum amount of zinc needed for full activity—2 g atoms of zinc per mole of protein—there appears to be no binding of halide ions to the protein-bound zinc ions. In contrast, when there is a high metal-enzyme ratio, a large relaxation enhancement is observed, demonstrating coordination of halide ions to the metal ions.Addition of inorganic phosphate causes no change in the ³⁵Cl^? relaxation in the presence of metal-free enzyme. However, marked decreases in relaxation are observed upon addition of phosphate to the Zn²⁺-alkaline phosphatase. The relaxation measurements carried out in the presence of phosphate show that substrate binding does prove to be metal-ion dependent. Furthermore, experiments with inorganic phosphate suggest the tight binding of one phosphate to the alkaline phosphatase.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.     

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca²⁺ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca²⁺ with Ba²⁺, Mn²⁺, Mg²⁺ and Sr²⁺ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe²⁺ and Zn²⁺ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg²⁺, Sn²⁺ and Sr²⁺, respectively. On replacing Ca²⁺ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg²⁺ and Sr²⁺, respectively. Only Ca²⁺ resulted in a marked increase in the melting temperature (T_m) of the enzyme by 12-21 °C, while Ba²⁺, Mn²⁺, Sr²⁺ or Cu²⁺ resulted in a modest (2-3.5 °C) increase in T_m. In the presence of 1-5 mM Ca²⁺, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.     

Complexation of NADH analogs with divalent metal ions: Dependence on the 3-substituent of 1-benzyl-1,4-dihydropyridines

In light of the critical role of divalent metal ions in the chemistry of coenzyme NADH analogs, complexation of 1-benzyl-3-substituted(X)-1,4-dihydropyridines (1, X=CONH₂; 2, X=CSNH₂; 3, X=COOCH₃; 4, X=COCH₃) with divalent metal ions (Mg²⁺, Zn²⁺, and Co²⁺) in dry acetonitrile was studied spectroscopically and kinetically. Presence of the metal ions causes red-shift of absorption band of NADH analogs and the rate retardation for the reaction between NADH analogs and N-methylacridinium ion. Analysis of the spectroscopic and kinetic data indicates that the NADH analogs form 1 : 1 complexes with the metal ions. The decreasing order of the magnitude of the association constants, K, is 1 2 4 3 for a given metal ion, and Mg²⁺ Zn²⁺ > Co²⁺ for a given NADH analog. The results strongly suggest that the primary binding site for the metal ions is the carbonyl oxygen (or thiocarbonyl sulfur) of the 3-substituent and that the amide nitrogen atom of the 3-substituent of 1 and 2 also ligates the metal ions, forming a bidentate structure and providing extra stability to the complexes of 1 and 2. Inhibition of reaction between NADH analogs and N-methylacridinium ion by the metal ions is attributed to inaccessibility of N-methylacridinium ion to the NADH analogs complexed with metal ions due to electrostatic repulsion.     

Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp⁵¹. The D153E enzyme had an increased k_cat in the presence of high concentrations of Mg²⁺, along with a decreased Mg²⁺ affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn₁ site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn²⁺, dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.     

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⁺.     

Effects of Magnesium Ions on Thermal Inactivation of Alkaline Phosphatase

The effect of Mg²⁺ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures and Mg²⁺ concentrations. Increasing the Mg²⁺ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the increased free Mg²⁺ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg²⁺ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence emission spectra showed that the emission maximum for Mg²⁺-containing system was always higher than that of Mg²⁺-free system, and the higher temperature enhanced this difference. In addition, Mg²⁺also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed.     

Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium

The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn²⁺, Mg²⁺, Co²⁺, and Zn²⁺ shift the optimal pH of phosphatase activity to 6.5 while Be²⁺ and Ca²⁺ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me²⁺ ions, Mn²⁺, Co²⁺, Zn²⁺, and Fe²⁺ decreased the K_m of histidinol phosphate at pH 6.5 from 5.5 mm (without Me²⁺) to 0.14 mm. Ni²⁺ and Be²⁺ increased the K_m to 22.2 and 25.0 mm, respectively, and Ca²⁺ and Mg²⁺ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn²⁺. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn²⁺ to the enzyme before the addition of the substrate, histidinol phosphate. A k_imn²⁺ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me²⁺ ions, Mg²⁺ being the most efficient followed by Mn²⁺. Prolonged incubation with EDTA led to irreversible inactivation.     

The relation between activity and zinc and chloride binding of Escherichia coli alkaline phosphatase.

The relation between Zn²⁺ binding of E. coli alkaline phosphatase and enzymatic activity and anion binding (using ³⁵Cl NMR) has been investigated. The results suggest the existence of two forms of the enzyme with different zinc binding properties. The anion binding associated with the enzyme's function appears to be an amino acid residue and not the Zn²⁺ ions; furthermore, there is a rapid internal motion at the anion binding site. ³⁵Cl relaxation studies in the presence of Mg²⁺ ions point to a marked interdependence of Mg²⁺ and Zn²⁺ binding.     

Cloning of an alkaline phosphatase gene from the moderately thermophilic bacterium<Emphasis Type="Italic"> Meiothermus ruber</Emphasis> and characterization of the recombinant enzyme

A gene that codes for an alkaline phosphatase was cloned from the thermophilic bacterium Meiothermus ruber, and its nucleotide sequence was determined. The deduced amino acid sequence indicates that the enzyme precursor including the putative signal sequence is composed of 503 amino acid residues and has an estimated molecular mass of 54,229 Da. Comparison of the peptide sequence with that of the prototype alkaline phosphatase from Escherichia coli revealed conservation of the regions in the vicinity of the corresponding phosphorylation site and metal binding sites. The protein was expressed in E. coli and its enzymatic properties were characterized. In the absence of exogenously added metal ions, activity was negligible; to obtain maximal activity, addition of free Mg²⁺ ions was required. Zn²⁺ ions had an inhibitory effect on the activity of the M. ruber enzyme. The pH and temperature optima for activity were found to be 11.0 and 62°C, respectively. The enzyme was moderately thermostable: it retained about 50% activity after incubation for 6 h at 60°C, whereas at 80°C it was completely inactivated within 2 h. The Michaelis constant for cleavage of 4-nitrophenylphosphate was 0.055 mM. While having much in common with other alkaline phosphatases, the M. ruber enzyme presents some unique features, such as a very narrow pH range for activity and an absolute requirement for magnesium for activity.Communicated by G. P. Georgiev     

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg²⁺ for activity and binds three to four ions tightly in the absence of ligands: K_D = 0.8 μM for one ion with a K_D of 38 μM for the other Mg²⁺ ions. However, the enzyme requires 5–10 mM Mg²⁺ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K_D for Mg²⁺; D26A, E39A, and E41A showed no significant change in the Mg²⁺ requirement for optimal activity. D38A also showed an increased K_m, but little effect on k_cat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg²⁺ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC₅₀～250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li⁺ with an IC₅₀ of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.     

Inhibitory Effect of Magnesium Ion on the Human Placental Alkaline Phosphatase-Catalyzed Reaction in a Reverse Micellar System

Human placental alkaline phosphatase is a membrane-anchored protein. Entrapping the enzyme into a reverse micellar vesicle mimics the in vivo conditions and allows examination of the properties of the enzyme. Placental alkaline phosphatase is enzymatically active in Aerosol-OT/isooctane reverse micelles. Substantially different kinetic behavior of the enzyme has been observed in aqueous or reverse micellar systems. In aqueous solution, Mg²⁺ is a nonessential activator of the enzyme. In the experiments described in the present report Mg²⁺ was found to be an inhibitor for the enzyme in reverse micelles. This inhibition is presumably due to a time-dependent conformational change of the enzyme molecule, which resulted in a curvature in the recorder tracings of the enzyme assays. The Mg²⁺-induced conformational change of the enzyme was completely prevented by phosphate and partially reserved by EDTA. High concentrations of Zn²⁺ also strongly inhibited enzyme activity in both aqueous and reverse micellar solvent systems, presumably by occupying the Mg²⁺ (M3) site of the enzyme. However, binding of Zn²⁺ at the M3 site did not cause conformational change of the enzyme and the enzyme assay tracing was linear. The M3 site of the enzyme is proposed to have a modulatory role in vivo using magnesium ion as the modulator.     

Substrate-dependent metal preference of PPM1H, a cancer-associated protein phosphatase 2C: comparison with other family members

Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn²⁺ when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg²⁺ is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is always closer to that with Mn²⁺ alone, irrespective of the substrate. This preponderance of Mn²⁺ is explained by its greater affinity for PPM1H than Mg²⁺. From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals, in relation to the substrate, may be responsible for this intriguing property.     

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg²⁺ as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg²⁺ concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n_H) is 2.0 ± 0.1, indicative of the involvement of two Mg²⁺ ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC₅₀ for the compound is 14 μM for the Mg²⁺-supported reaction, and 8 μM for the Mn²⁺-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism.     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

KpnI REase recognizes palindromic sequence, GGTAC↓C, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg²⁺. Surprisingly, Ca²⁺ suppresses the Mg²⁺-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca²⁺-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg²⁺- and Mn²⁺-mediated reactions and was about three times slower with Ca²⁺. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg²⁺-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity in its DNA recognition and cleavage properties.     

Designing a Highly Efficient Refolding System for Alkaline Phosphatase using Combination of Cyclodextrin and Mg<Superscript>2+</Superscript> Ion

Two different folding aids including α-cyclodextrin and Mg²⁺ ions were applied to alkaline phosphatase refolding. The refolding yield depending on concentration of each of the refolding agents reached to 55 and 52% in the presence of 100 mM α-cyclodextrin and/or 5 mM Mg²⁺ ions, respectively. However, the refolding yield, mediated by combination of α-cyclodextrin and Mg²⁺ ions, was more than 96%. Replacement of Mg²⁺ ion with other type of ions which interact with α-cyclodextrin interfere with the function of cyclodextrin resulting in low refolding yields.