Short unligated sticky ends enable the observation of circularised DNA by atomic force and electron microscopies.

A comparative study of the stabilisation of DNA sticky ends by divalent cations was carried out by atomic force microscopy (AFM), electron microscopy and agarose gel electrophoresis. At room temperature, molecules bearing such extremities are immediately oligomerised or circularised by addition of Mg2+or Ca2+. This phenomenon, more clearly detected by AFM, requires the presence of uranyl salt, which stabilises the structures induced by Mg2+or Ca2+. DNA fragments were obtained by restriction enzymes producing sticky ends of 2 or 4 nucleotides (nt) in length with different guanine plus cytosine (GC) contents. The stability of the pairing is high when ends of 4 nt display a 100% GC-content. In that case, 95% of DNA fragments are maintained circular by the divalent cations, although 2 nt GC-sticky ends are sufficient for a stable pairing. DNA fragments with one blunt end and the other sticky appear as dimers in the presence of Mg2+. Dimerisation was analysed by varying the lengths and concentrations of DNA fragments, the base composition of the sticky ends, and also the temperature. Our observation provides a new powerful tool for construction of inverted dimers, and circularisation, ligation analysis or short bases sequence interaction studies.     

The divalent cations Ca2+ and Mg2+ play specific roles in stabilizing histone-DNA interactions within nucleosomes that are partially redundant with the core histone tail domains

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational repositioning upon removal of the core histone tail domains under physiological conditions, indicating that the tails influence the choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a nonbiased chemical cleavage reagent, indicative of an increase in the motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg(2+) and Ca(2+) can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict repositioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg(2+) after tail removal, the original distribution of translational positions was not re-established, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn(2+), Fe(2+), Co(2+), and Mn(2+) had little or no effect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg(2+) and Ca(2+) ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.     

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

DNase Induced After Infection of KB Cells by Herpes Simplex Virus Type 1 or Type 2 II. Characterization of an Associated Endonuclease Activity

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.     

Divalent cations stimulate preferential recognition of a viral DNA end by HIV-1 integrase.

In the presence of a divalent metal cofactor (Mg2+ or Mn2+), retroviral-encoded integrase (IN) catalyzes two distinct reactions: site-specific cleavage of two nucleotides from both 3' ends of viral DNA, and sequence-independent joining of the recessed viral ends to staggered phosphates in a target DNA. Here we investigate human immunodeficiency virus type 1 (HIV-1) IN-DNA interactions using surface plasmon resonance. The results show that IN forms tight complexes both with duplex oligonucleotides that represent the viral DNA ends and with duplex oligonucleotides with an unrelated sequence that represent a target DNA substrate. The IN-DNA complexes are stable in 4.0 M NaCl, or 50% (v/v) methanol, but they are not resistant to low concentrations of SDS, indicating that their stability is highly dependent on structural features of the protein. Divalent metal cofactors exert two distinct effects on the IN-DNA interaction. Mn2+ inhibits IN binding to a model target DNA with the apparent Kd increasing approximately 3-fold in the presence of this cation. On the other hand, Mn2+ (or Mg2+) stimulates the binding of IN to a model viral DNA end, decreasing the apparent Kd of this IN-viral DNA complex approximately 6-fold. Such metal-mediated stimulation of the binding of IN to the viral DNA is totally abolished by substitution of the subterminal conserved CA/GT bp with a GT/CA bp, and is greatly diminished when the viral DNA end is recessed or "pre-processed." IN binds to a viral duplex oligonucleotide whose end was extended with nonviral sequences with kinetics similar to the nonviral model target DNA. This suggests that IN can distinguish the integrated DNA product from the viral donor DNA in the presence of divalent metal ion. Thus, our results show that preferential recognition of viral DNA by HIV-1 IN is achieved only in the presence of metal cofactor, and requires a free, wild-type viral DNA end.     

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (> or =2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Role of divalent metals in the activation and regulation of insulin receptor tyrosine kinase

Multiple equilibrium equations were solved to separate the individual effects of ionic divalent metals, free nucleotides and their chelated species on insulin receptor tyrosine kinase (IRTK). Basal IRTK is activated by divalent metal cations when present in excess of that required for substrate formation, indicating the presence of a divalent cation-dependent regulatory site on the kinase. The activatory order for basal activity was Mn2+ greater than Co2+ greater than Mg2+ and Ca2+ = 0. The insulin-dependent activation of IRTK was minimal in the absence of excess free divalent metal, even when the concentration of MnATP or MgATP substrate present exceeded the apparent Km of the kinase. The activatory order for insulin-dependent activation of IRTK changed to Mg2+ greater than Mn2+ and Co2+ = 0. The titration of the MnCl2 saturation response at several concentrations of MgCl2 revealed that the insulin-dependent response of IRTK increases as a function of increasing MgCl2, while basal activity was unaffected. This enhancement of the responsiveness to insulin in the presence of both cations was not due to differing affinities of the kinase for substrate, as evidenced by nearly identical apparent Km values for MnATP and MgATP. The Mg2+-dependent increase in the response of the kinase to insulin may be due to Mg2+ inducing a stronger coupling between receptor and kinase than that observed with Mn2+ alone. The plotting of the effect of several concentrations of free divalent metals on substrate saturation curves revealed that an increase in either of the reactants increased the affinity of the insulin-activated kinase for the other respective reactant. Accordingly, free divalent metal and metal-ATP substrate interact with IRTK in a mutually inclusive manner. CaCl2 saturation curves in the presence of constant MnCl2 and increasing MgCl2 showed that the affinity of IRTK for Ca2+ decreases and the affinity for CaATP increased with increasing Mg2+. Our data suggests that IRTK contains three sites for interaction with divalent metal cations: a MeATP (active) site, a regulatory site, and a metal-dependent site acting to couple the receptor with the kinase.     

Binding of Mg2+, Cd2+, and Ni2+ to liquid crystalline NaDNA: polarized light microscopy and NMR investigations

The interaction of the divalent metal ions Mg(2+), Cd(2+), and Ni(2+) with liquid crystalline NaDNA solutions (molar ratios Me(2+)/DNA-phosphate 相似文献   

Mycobacterium tuberculosis isocitrate lyase (MtbIcl): role of divalent cations in modulation of functional and structural properties

Isocitrate lyase (Icl), an enzyme that plays an important role in the regulation of isocitrate flux and anaplerotic replenishment of pool of substrate required for biosynthetic process in Mycobacterium tuberculosis is a potential drug target for the antituberculosis drugs. Divalent cations induce differential effect of activation and inhibition of MtbIcl functional activity. The study for the first time demonstrates that interaction of cations with MtbIcl results in differential modulation of the enzyme structure which is probably the underlying mechanism for differential modulation of functional activity of enzyme by divalent cations. The Mg(2+) and Mn(2+) ions act as activators of the enzyme and in their absence no enzymatic activity was observed. These cations do not induce any significant structural alteration in the enzyme as observed by far-UV CD and solvent denaturation studies using chaotropic salts. However, the thermal denaturation studies demonstrate that they do interact with the noncatalytic alpha/beta barrel core domain of the enzyme and destabilize it. The inhibitors Zn(2+) and Cd(2+) interact directly with the catalytic domain of the enzyme and unfold it as a result of which complete loss of the enzymatic activity is observed in their presence. The results obtained from the studies provide intriguing insight into the possible mechanism of divalent cation-induced changes in structure, function, and stability of MtbIcl.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd.

Interactions of divalent metal cations (Mg2+, Ca2+, Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) with DNA have been investigated by laser Raman spectroscopy. Both genomic calf-thymus DNA (> 23 kilobase pairs) and mononucleosomal fragments (160 base pairs) were employed as targets of metal interaction in solutions containing 5 weight-% DNA and metal:phosphate molar ratios of 0.6:1. Raman difference spectra reveal that transition metal cations (Mn2+, Co2+, Ni2+, Cu2+, Pd2+, and Cd2+) induce the greatest structural changes in B-DNA. The Raman (vibrational) band differences are extensive and indicate partial disordering of the B-form backbone, reduction in base stacking, reduction in base pairing, and specific metal interaction with acceptor sites on the purine (N7) and pyrimidine (N3) rings. Many of the observed spectral changes parallel those accompanying thermal denaturation of B-DNA and suggest that the metals link the bases of denatured DNA. While exocyclic carbonyls of dT, dG, and dC may stabilize metal ligation, correlation plots show that perturbations of the carbonyls are mainly a consequence of metal-induced denaturation of the double helix. Transition metal interactions with the DNA phosphates are weak in comparison to interactions with the bases, except in the case of Cu2+, which strongly perturbs both base and phosphate group vibrations. On the other hand, the Raman signature of B-DNA is largely unperturbed by Mg2+, Ca2+, Sr2+, and Ba2+, suggesting much weaker interactions of the alkaline earth metals with both base and phosphate sites. A notable exception is a moderate perturbation by alkaline earths of purine N7 sites in 160-base pair DNA, with Ca2+ causing the greatest effect. Correlation plots demonstrate a strong interrelationship between perturbations of Raman bands assigned to ring vibrations of the bases and those of bands assigned to exocyclic carbonyls and backbone phosphodiester groups. However, strong correlations do not occur between the Raman phosphodioxy band (centered near 1092 cm-1) and other Raman bands, suggesting that the former is not highly sensitive to the structural changes induced by divalent metal cations. The structural perturbations induced by divalent cations are much greater for > 23-kilobase pair DNA than for 160-base pair DNA, as evidenced by both the Raman difference spectra and the tendency toward the formation of insoluble aggregates. In the presence of transition metals, aggregation of high-molecular-weight DNA is evident at temperatures as low as 11 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59.The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA.We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.     

Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule.

The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis. The DNA contains six EcoRI cleavage sites. Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA. In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers. No unit-length linear structures were detected. The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease. On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

Direct transfer of Ku between DNA molecules with nonhomologous ends.

The Ku protein is an essential protein for DNA double-strand-break repair by the pathway of nonhomologous DNA end-joining (NHEJ). A previous study showed that Ku bound to one DNA molecule could transfer directly to another DNA molecule without being released into the solution first. Direct transfer requires the two DNA molecules having homologous cohesive ends with a minimum of four complementary bases. Results of this study reveal that direct transfer activity of Ku is regulated by NaCl and MgCl2. Increasing either one of the two cations can decrease the required amount of the other. However, the DNA end-binding activity of Ku is not affected by changing the concentration of the cations, indicating that the two activities are regulated independently. Most importantly, the results also show that Ku can transfer directly from one DNA molecule to another one with nonhomologous ends under the condition of 200 mM NaCl and 5mM MgCl2. The ability of direct transfer between DNAs with nonhomologous ends suggests that Ku can align or juxtapose two DNA ends during NHEJ.     

Neutralization of acidic residues in helix II stabilizes the folded conformation of acyl carrier protein and variably alters its function with different enzymes

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

EcoRV restrictase: physical and catalytic properties of homogenous enzyme

With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.     

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.