Mg2+ and Ni2+ ion effect on stability and structure of triple poly I.poly A.poly I helix

The effects of Mg2+ and Ni2+ ions on the absorption spectra of IMP, single-stranded poly I and three-stranded A2I in solutions with 0.1 M Na+ (pH 7) have been studied. In contrast to Mg2+ ions, the Ni2+ ions affect the absorption spectra of these polynucleotides and IMP. The concentration dependences of the intensity at the extrema in the differential UV spectra suggest that in the region of high Ni2+ concentrations ionic complexes with poly I and A2I are formed, which are characterized by the association constants K'I = 2000 M(-1) and K'A2I = 550 M(-1), respectively. The shape of the DUV spectra prompts the conclusion that these complexes are formed due to the inner-sphere interaction of Ni2+ ions with N7 of poly I and A2I presumably due to the outer-sphere Ni2+-O6 interaction. The formation of the complexes leads to destruction of A2I triplexes. The dependences of the melting temperature (T(m)) of A2I on Mg2+ and Ni2+ concentrations have been measured. The thermal stability is observed to increase at the ionic contents up to 0.01 M Mg2+ and only to 2x10(-4) M Ni2+. At higher contents of Ni2+ ions, T(m) lowers and the cooperativity of A2I melting decreases continuously. In all the cases the melting process is the A2I-->A+I+I (3-->1) transition. According to the "ligand" theory, these effects are generated by the energy-advantageous Ni2+ binding to single-stranded poly I (K'A2I < K'I) and by the greater number of binding sites which appears during the 3-->1 transition and is entropy-advantageous.     

Distribution of Mn2+ ions around poly(rI).poly(rC)

The distribution of bound Mn²⁺ ions about poly(rI)·poly(rC) has been studied by measuring the effect of this paramagnetic metal ion on the relaxation behavior of poly(rI)·poly(rC) protons. By combining selective spin – lattice and spin – spin relaxation rates for various protons, some of the principle regions of ion association can be identified. The relaxation data on the CH6 proton are consistent with a < 10% occupancy of phosphate inner-sphere binding sites. The broadening of the imino proton resonance requires a substantial occupancy of sites located in the major groove, possibly near IN7. This would also be consistent with the observation that IH8 resonance is the proton most susceptible to relaxation by Mn²⁺. The relaxation data for the IH2 proton indicate a relatively low occupancy of minor-groove binding sites (e.g., IN3).     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.     

Interaction of metal ions with polynucleotides and related compounds. IX. Unwinding and rewinding of poly(A + U) and poly(I + C) by copper(II) ions

It is demonstrated that, poly(A + U) and poly(I + C) are both formed under low ionic strength conditions. Continuous variation studies indicate the formation of copper(II) complexes of poly A, poly C, and poly I, but not of poly U. Copper(II) in a 1:1 ratio to polynucleotide prevents the formation of poly(A + U) and brings about the dissociation of the poly (A + U) complex produced in the absence of the metal. Poly (I + C) is similarly dissociated by copper(II) ions. The addition of sufficient electrolyte reverses the copper(II) induced dissociation of poly(I + C). The effect of copper(II) on ordered synthetic polynucleotides is thus very similar to its effect on DNA.     

Anticodon loop of tRNAPhe: structure, dynamics, and Mg2+ binding

The structure, dynamics, and Mg2+ binding reactions of the isolated anticodon hairpin loop from tRNAPhe (yeast) have been analyzed by fluorescence-detected temperature-jump relaxation, melting experiments, and equilibrium sedimentation. Most of the measurements were performed at an ionic strength of 0.15 M and at temperatures below 25 degrees C, where the hairpin loop proved to be stable. A relaxation effect with a time constant of approximately 100 microseconds, indicated by the Wye base fluorescence, is attributed to a conformational change of the anticodon loop and is very similar to a corresponding transition observed previously for the whole tRNAPhe molecule. A Mg2+ binding site reflected by an inner-sphere relaxation process and associated with a strong increase of the Wye base fluorescence closely resembles a corresponding site observed in the complete tRNAPhe and is attributed to a site in the anticodon loop identified by X-ray analysis. In addition to the Mg2+ site in the loop, which is associated with a binding constant of 2 X 10(3) M-1, the existence of sites with a higher affinity is demonstrated by an unusual relaxation effect, showing a minimum in the reciprocal time constant with increasing Mg2+ concentration. The experimental data can be described by a transition between two states and Mg2+ binding to both states resulting in a reaction cycle, which is extended by an additional Mg2+ binding reaction to one of the states. The unusual effect has not been observed for the complete tRNAPhe and is also not observed when Ca2+ is added instead of Mg2+. This result indicates the existence of a conformational change involving Mg2+ inner-sphere complexation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state properties of calcium binding to parvalbumins from bullfrog skeletal muscle: effects of Mg2+, pH, ionic strength, and temperature

To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I = 0.106, pH 6.80, at 20 degrees C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, KCa = 1 X 10(7) M-1, KMg = 900 M-1; for PA-2, KCa = 6 X 10(6) M-1, KMg = 830 M-1 (I = 0.106, pH 6.80, at 20 degrees C). The apparent binding constants are strongly dependent on temperature: at 10 degrees C for PA-1, KCa = 2 X 10(8) M-1, KMg = 10(4) M-1; for PA-2, KCa = 5 X 10(7) M-1, KMg = 5 X 10(3) M-1 (I = 0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.     

Rat ovarian and adrenal prolactin receptors. Sizes and effects of divalent metal ions

Receptor fractions were prepared from follicle-rich ovaries (for FSH), luteal cell-rich ovaries (for LH and PRL), and adrenals (for PRL) of rats. Divalent metal ions, Mg++, Ca++, and Mn++ showed inhibitory effects on the binding of LH and FSH to their receptors. The binding of the former was more sensitive to these ions than the latter. On the other hand they showed bell-shaped promotive effects on PRL-ovarian receptor binding, the maximal effects being observed at 10-20 mM. Besides these ions, Ba++ also had a promotive effect, while other divalent metal ions such as Zn++, Cd++, Ni++, and Co++ showed inhibitory effects on PRL-ovarian receptor binding at 5 mM. Mg++ and Ca++ also promoted PRL-adrenal receptor binding, while Mn++ promoted the binding at 10 mM but inhibited it at higher concentrations. Association constant (Ka) and binding capacity (Bmax) of PRL receptors of the ovary and the adrenal were significantly different (ovary: Ka = 0.69 X 10(10) M-1, Bmax = 62 fmol/mg protein, adrenal: Ka = 0.21 X 10(10) M-1, Bmax = 99 fmol/mg protein). Ka of the ovarian PRL receptor was not influenced by these divalent ions, while that of the adrenal receptor was doubled by Ca and Mn ions, Bmax of the latter was also increased. A cooperative effect of Mg and Ca ions was observed on Ka and Bmax of the adrenal receptor. The sizes of the PRL binding sites of these organs revealed by affinity labelling were 17K and 40K in the ovary, and 40K and 110K in the adrenal. These results indicate the different properties of receptors in these different target organs.     

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.     

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.     

Octahedral metal coordination in the active site of glyoxalase I as evidenced by the properties of Co(II)-glyoxalase I

Co(II)-glyoxalase I has been prepared by reactivation of apoenzyme from human erythrocytes with Co2+. The visible absorption spectrum showed maxima at 493 and 515 nm and shoulders at 465 and 615 nm. The absorption coefficients at 493 and 515 nm were 35 and 33 M-1 cm-1/cobalt ion, respectively; i.e. 70 and 66 M-1 cm-1 for the dimeric metalloprotein. The product of the enzymatic reaction, S-D-lactoylglutathione, although binding to Co(II)-glyoxalase I, had no demonstrable effect on the visible absorption spectrum, indicating binding outside the first coordination sphere of the metal. The EPR spectrum at 3.9 K was characterized by g1 approximately 6.6, g2 approximately 3.0, and g3 approximately 2.5, and eight hyperfine lines with A1 = 0.025 cm-1. Binding of the strong competitive inhibitor S-p-bromobenzylglutathione to Co(II)-glyoxalase I gave three g values: 6.3, 3.4, and 2.5, indicating a conformational change affecting the environment of the metal ion. Both optical and EPR spectra strongly suggest a high spin Co2+ with octahedral coordination in the active site of the enzyme. The similarities in kinetic properties between native Zn(II)-glyoxalase I and enzyme substituted with Mg2+, Mn2+, or Co2+ is consistent with the view that these enzyme forms have the same metal coordination in the protein.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

Acoustical investigation of poly(dA).poly(dT), poly[d(A-T)], poly(A).poly(U) and DNA hydration in dilute aqueous solutions.

Apparent molar adiabatic compressibilities and apparent molar volumes of poly[d(A-T)].poly[d(A-T)], poly(dA).poly(dT), DNA and poly(A).poly(U) in aqueous solutions were determined at 1 degree C. The change of concentration increment of the ultrasonic velocity upon replacing counter ion Cs+ by the Mg2+ ion was also determined for these polymers. The following conclusions have been made: (1) the hydration of the double helix of poly(dA).poly(dT) is remarkably larger than that of other polynucleotides; (2) the hydration of the AT pair in the B-form DNA is larger than that of the GC pair; (3) the substitution of Cs+ for Mg2+ ions as counter ions results in a decrease of hydration of the system polynucleotide plus Mg2+, and (4) the magnitude of this dehydration depends on the nucleotide sequence; the following rule is true: the lesser is a polynucleotide hydration, the larger dehydration upon changing Cs+ for Mg2+ ions in the ionic atmosphere of polynucleotide.     

Spectral properties of Co(II)- and Ni(II)-activated rabbit muscle pyruvate kinase.

Stoichiometry, kinetics, and optical properties of rabbit muscle pyruvate kinase activated with Co(II), Ni(II), Mg(II), and Mn(II) were studied. The stoichiometry of metal binding to enzyme was found to be 4 metal ions per tetrameric enzyme for Co(II) and Ni(II) by carrying out circular dichroic titrations. Cu(II) and Fe(II) were inactive. Ca(II) and Zn(II) were not activating, and were inhibitory with respect to all of the active cations. The temperature dependence of the optimal velocity is similar for all activating metals. The pH rate profiles suggest that there are two classes of enzyme activation by metal ions. Mg(II) and Mn(II) are quite similar to each other while Co(II) and Ni(II) are different from them but similar to each other. Absorption, natural, and magnetic CD in the visible region were used to probe the environment of the activating divalent cation in Ni(II)- and Co(II)-activated pyruvate kinase and their complexes with substrates and inhibitors...     

Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.     

Mechanism of codon recognition by transfer RNA and codon-induced tRNA association

The steps of UUC recognition by tRNAPhe were analysed by temperature-jump measurements. At ion concentrations close to physiological conditions we found three relaxation processes, which we assigned to (1) formation of codon-anticodon complexes, (2) a conformational change of the anticodon loop coupled with Mg2+ binding, and (3) codon-induced association of tRNA. The relaxation data were evaluated both by the usual procedure (fitting the exponentials evaluated from the individual experiments of a set to a reaction model) and by "global fitting", i.e. fitting a set of relaxation curves obtained at various concentrations directly to a reaction model, thus leaving out the intermediate exponential fitting step. The data can be represented quantitatively by a three-step model: the codon binds to the anticodon at a rate of 4 X 10(6) to 6 X 10(6) M-1S-1 as is usual for the formation of oligomer helices; the conformation change of the anticodon loop is associated with inner sphere complexation of Mg2+ at a rate of 10(3) S-1; the codon-tRNA complexes form dimers at a rate of 5 X 10(6) to 15 X 10(6) M-1S-1. A similar mechanism is found for the binding of the wobble codon UUU to tRNAPhe at increased concentrations of Mg2+. Measurements at different Mg2+ concentrations demonstrate the distinct role of this ion in the codon recognition and the codon-induced tRNA dimerization. We propose a simple mechanism, based upon the special properties of magnesium ions, for long-distance transfer of reaction signals along nucleic acid chains.     

Interaction of transition metal ions with Z form poly d(A-C).poly d(G-T) and poly d(A-T) studied by I.R. spectroscopy.

Interactions between Ni2+, Co2+ and purine bases have been studied by I.R. spectroscopy in the case of double stranded regularly alternating purine-pyrimidine polynucleotides poly d(A-T), poly d(A-C).poly d(G-T) and poly d(G-C). The spectra of polynucleotide films have been recorded in hydration and salt content conditions which correspond to the obtention of the classical right-handed (A,B) and left-handed (Z) helical conformations. Selective deuteration of the 8C site of purines has been obtained and is used to detect interactions between the transition metal ions and the adenine or guanine bases. The spectral region between 1500 and 1250 cm-1 corresponding to base in-plane vibrations and involving also the glycosidic linkage torsion is discussed in detail. The selective interaction between the transition metal ion and the 7N site of the purine base is considered to be partly responsible for the stabilization of the base in a syn conformation, which favours the adoption by the polynucleotide (poly d(G-C), poly d(A-C).poly d(G-T) or poly d(A-T)) of a Z type conformation.     

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.