Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.     

Comparison of rate constants for (PO3-) transfer by the Mg(II), Cd(II), and Li(I) forms of phosphoglucomutase

Net rate constants that define the steady-state rate through a sequence of steps and the corresponding effective energy barriers for two (PO3-)-transfer steps in the phosphoglucomutase reaction were compared as a function of metal ion, M, where M = Mg2+ and Cd2+. These steps involve the reaction of either the 1-phosphate or the 6-phosphate of glucose 1,6-bisphosphate (Glc-P2) bound to the dephosphoenzyme (ED) to produce the phosphoenzyme (EP) and the free monophosphates, glucose 1-phosphate (Glc-1-P) or glucose 6-phosphate (Glc-6-P): EP.M + Glc-1-P----ED.M.Glc-P2----EP.M.Glc-6-P6. Before this comparison was made, net rate constants for the Cd2+ enzyme, obtained at high enzyme concentration via 31P NMR saturation-transfer studies [Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry (preceding paper in this issue)], were appropriately scaled by using the observed constants to calculate both the expected isotope-transfer rate at equilibrium and the steady-state rate under initial velocity conditions and comparing the calculated values with those measured in dilute solution. For the Mg2+ enzyme, narrow limits on possible values of the corresponding net rate constants were imposed on the basis of initial velocity rate constants for the forward and reverse directions plus values for the equilibrium distribution of central complexes, since direct measurement is not feasible. The effective energy barriers for both the Mg2+ and Cd2+ enzymes, calculated from the respective net rate constants, together with previously values for the equilibrium distribution of complexes in both enzymic systems [Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018-4025], show that the 100-fold decrease in the kappa cat for the Cd2+ relative to the Mg2+ enzyme is caused by two factors: the increased stability of the intermediate bisphosphate complex and the decreased ability to cope with the phosphate ester involving the 1-hydroxyl group of the glucose ring. In fact, it is unlikely that the efficiency of (PO3-) transfer to the 6-hydroxyl group of bound Glc-1-P (thermodynamically favorable direction) is reduced by more than an order of magnitude in the Cd2+ enzyme. By contrast, the efficiency of the Li+ enzyme in the same (PO3-)-transfer step is less than 4 x 10(-8) that of the Mg2+ enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The synthesis and characterisation of polyene complexes with divalent metal ions: Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II)

Metal ion (Mg(II), Ca(II), Zn(II), Cu(II), Ni(II)) complexes of nystatin and amphotericin B (polyene antibiotics) have been prepared as solids. The stoichiometry of the complexes has been established. IR, ESR investigation indicates the metal-ligating sites in the polyene molecules. The existence of such complexes is discussed in the light of polyene mode- of-action theories.     

Stopped-flow kinetic studies of Ca(II) and Mg(II) dissociation in cod parvalbumin and bovine alpha-lactalbumin

The dissociation kinetics of complexes of bovine alpha-lactalbumin and cod parvalbumin with Ca(II) and Mg(II) ions induced by mixing of a Ca(II)- or MG(II)-loaded protein with a chelator of divalent cations (EDTA or EGTA) have been studied by means of the stopped-flow method with intrinsic protein fluorescence registration. Within the temperature interval from 10 to approx. 37 degrees C kinetic curves for Ca(II) removal from alpha-lactalbumin are monoexponential with a rate constant ranging from 0.006 to 1 s. Taking into account the rather low rate of fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca(II) ion from the protein and not a conformational transition which occurs after Ca(II) dissociation. At temperatures above 37 degrees C the kinetic curves require at least two exponential terms for a satisfactory fit. The second exponential seems to be due to denaturation of the apo form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants for Mg(II) bound to alpha-lactalbumin practically coincide with those for Ca(II). Within the temperature interval 10-30 degrees C the kinetic curves for Ca(II) and Mg(II) removal from parvalbumin are best fitted by a sum of two exponential terms identified as arising from the dissociation of cations from the two binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The determination of the separation of tyrosine-99 and tyrosine-138 in calmodulin: Radiationless energy transfer

The average separation of the phenolic groups of tyrosine-99 and tyrosine-138 has been measured by radiationless energy transfer between each tyrosine and the nitro derivative of the second tyrosine. A separation of 16.7 ± 0.7 Å was found in the absence of Ca²⁺ and 15.5 ± 0.7 Å in the presence of Ca²⁺.     

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.     

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.     

Microcalorimetric evaluation of the biological activity of polyene complexes with divalent metal ions: Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II)

Polyene complexes with Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II) have been prepared and evaluated for biological activity in a flow microcalorimetric study. The bioactivities are all lower per g of complex than is the bioactivity of the patent polyene, nystatin. However extrapolation of the linear bioassay data suggests that because of enhanced solubilities the metal ion complexes may be able to yield higher overall bioactivity than can nystatin alone.     

Al3+ versus Ca2+ ion binding to methionine and tyrosine spin-labeled bovine brain calmodulin.

Bovine calmodulin analogues, spin-labeled at either methionine or tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies to investigate possible calmodulin interactions with aluminum ion. The study attempts to clarify a previous report in the literature (H. Siegel, R. Coughlin, and A. Haug, Biochem. Biophys. Res. Commun. 115, 512 (1983)) which indicated, on the basis of EPR experiments on methionine spin-labeled protein, significant interaction between calmodulin and aluminum ion at pH = 6.5. In EPR metal ion titration experiments we have found that the signal line-shape (from both methionine and tyrosine spin labels) changed dramatically with the addition of calcium ion, but was virtually unchanged with the addition of aluminum ion at pH = 6.5. Experiments performed at pH = 5.5, where significantly more "free" aluminum ion (i.e., Al(H2O)6(3+) = Al3+) is present, also failed to produce the line-narrowing effect observed in the earlier study. Based on our EPR experiments, in the pH range 5.5 to 6.5, we find no evidence for significant interaction between calmodulin and aluminum ion.     

Interaction of adenosine 5'-triphosphate with metal ions X-ray structure of ternary complexes containing Mg(II), Ca(II), Mn(II), Co(II), ATP and 2,2'-dipyridylamine

The X-ray structures of the isomorphous Mg2+, Ca2+, Mn2+ and Co2+ complexes of ATP have been determined. The metal ions are wrapped in hexa-coordination by the alpha, beta and gamma phosphate groups of two ATP molecules thus blocking the interaction of the metal ions with the adenine base. A second metal ion which is fully hydrated, M(H2O)2+(6), is engaged in a strong hydrogen bond with the gamma phosphate group of ATP and suggests a possible step in facilitating the cleavage between the beta and gamma phosphates in phosphoryl transfer reactions.     

Impact of Humin on Soil Adsorption and Remediation of Cd(II), Pb(II), and Cu(II)

In situ immobilization constitutes a promising technology for the mitigation of contaminants, through the reduction of metal bioavailability and mobility. This study investigated the adsorption isotherms and kinetic characteristics of humin extracted from peat soils. We also studied the influences of the pH, ionic strengths, and soluble organic matter concentrations of soil solutions on the adsorptive properties of humin, and compared its ability to detoxify potentially toxic metals in both actual and simulated soil solutions. The study results indicated that humin contains a massive population of oxygen-containing functional groups. Its adsorption capacity for Pb(II) was greater than that for Cu(II), which exceeded that for Cd(II). The adsorption of humin for Pb(II) conformed to the Freundlich model, while the adsorption of humin for Cd(II) and Cu(II) followed the Langmuir model. The adsorption kinetics of humin with respect to potentially toxic metals aligned well with second-order kinetics equations. As the pH was elevated, the potentially toxic metal adsorption by humin increased rapidly. Electrolyte ions and tartaric acids in solution both inhibited the adsorption of potentially toxic metals by humin, and its ability to inactivate potentially toxic metals. This was shown to be improved in actual field soil solutions in contrast to simulated soil solutions.     

Synthesis and characterization of Mg(II), Mn(II), Zn(II) and Cd(II) complexes with a new heptaaza Schiff base pendant-armed macrocycle: X-ray crystal structure, NMR and computational study

New 2-aminoethyl pendant-armed Schiff base macrocyclic complexes, [ML⁷]²⁺ (M = Mn(II), Mg(II), Zn(II) and Cd(II)), have been prepared via M(II) templated [1 + 1] cyclocondensation of 2,6-diacetylpyridine with a new branched hexamine, N,N,N′,N′-tetrakis(2-aminoethyl)-2,2-dimethylpropane-1,3-diamine. The ligand is a 16-membered pentaaza macrocycle having two 2-aminoethyl pendant arms [L⁷ is 2,14-dimethyl-6,10-bis(2-aminoethyl)-3,6,10,13,19-pentaazabicyclo[13.3.1]8,8-dimethylnonadeca-1(19),2,13,15,17-pentaene]. The crystal structures of [MnL⁷]²⁺ and [MgL⁷]²⁺ were determined from X-ray diffraction data. The geometry of the coordination sphere of complexes is a slightly distorted pentagonal bipyramid with the metal ion located within a pentaaza macrocycle and two pendant amines coordinating on opposite sides. All complexes were characterized by IR, microanalysis and except of [MnL⁷]²⁺ by ¹H NMR, ¹³C NMR, DEPT135, COSY(H, H) and HMQC spectroscopy. The data indicate that the structure is pentagonal bipyramidal in each case. The structure of all complexes has also been theoretically studied by ab initio Hartree-Fock and density functional theory methods.     

Manganese (II) And spin-labeled uridine 5'-diphosphate binding to bovine galactosyltransferase.

The kinetically observed Mn(II) activation as well as inhibition has been clarified for bovine galactosyltransferase. An electron spin resonance (ESR) titration of MnCl2 with galactosyltransferase alone at pH 8.0 clearly shows the existence of at least two metal ion binding sites with microscopic dissociation constants of 0.84 +/- 0.1 and 9.0 +/- 1.0 mM, respectively. The second site corresponds with either published kinetic constant for Mn(II) of 8.5 mM (inhibition) or 3.40 mM (activation). The contribution of the binary complex Mn(II)-UDPGal is of lesser significance, as concluded by its ESR measured Kdiss of 14.5 +/- 1.1 mM at pH 8.0. A spin-labeled inhibitor analog of UDPgalactose, UDP-4-O-(2,2,6,6-tetramethyl-4-piperidinyl-1-oxy), or UDP-R, was synthesized as a competitive inhibitor for UDPGal. It was shown from inhibition kinetics to be almost as potent an inhibitor as UDPGlu. The Ki values at pH 8.0 in the N-acetyllactosamine and lactose reactions were 0.38 +/- 0.04 and 0.63 +/- 0.06 mM, respectively, as compared with 0.10 +/- 0.01 and 0.094 +/- 0.009 mM for UDPGlu. An ESR titration of UDP-R with galactosyltransferase at pH 8.0 yielded direct physical dissociation constants of 0.40 +/- 0.07 and 0.53 +/- 0.08 mM in the absence and presence of alpha-lactalbumin, respectively. No other substrates (glucose of N-acetylglucosamine) nor Mn(II) were present.     

A family of polyamino phenolic macrocyclic ligands. Acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) ions

The acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) of four polyamino-phenol macrocycles 15-hydroxy-3,6,9-triazabicyclo[9.3.1]pentadeca-11,13,1¹⁵-triene L1, 18-hydroxy-3,6,9,12-tetraazabicyclo[12.3.1]octadeca-14,16,1¹⁸-triene L2, 21-hydroxy-3,6,9,12,15-pentaazabicyclo[15.3.1]enaicosa-17,19,1²¹-triene L3 and 24-hydroxy-3,6,9,12,15,18-hexaazabicyclo[18.3.1]tetraicosa-20,22,1²⁴-triene L4 are reported. The protonation and stability constants were determined by means of potentiometric measurements in 0.15 mol dm⁻³ NMe₄Cl aqueous solution at 298.1 K. L1 forms highly unsaturated Co(II), Cu(II), Zn(II) and Cd(II) mononuclear complexes that are prone to give dimeric dinuclear species with [(MH₋₁L1)₂]²⁺ stoichiometry, in solution. L2 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes that can coordinate external species as OH⁻ anion, giving hydroxylated complexes at alkaline pH. L3 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes and Co(II), Ni(II), Cu(II) and Zn(II) dinuclear [M₂H₋₁L3]³⁺ species. L4 forms stable mono- and dinuclear Co(II), Cu(II), Zn(II) and Cd(II) complexes, but only mononuclear species with Pb(II). The effect of macrocyclic size is considered in the discussion of results.     

Interaction of metal ions with humic-like models. Part VII. Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes of 2,5-dihydroxybenzoic acid

Complexes of formula M(2,5-DHB)₂4H₂O (M = Mn, Co, Ni, Zn, Cu and Cd; 2,5-DHB = 2,5-dihydroxybenzoate) were prepared and characterized by means of infrared and electronic spectroscopy, and by electron spin resonance. For the Zn complex the crystal and molecular structure was also determined by single-crystal X-ray diffraction analysis. The crystal is orthorhombic, space group Pbca (No. 61), with a = 18.503(4), b = 13.536(3), c = 6.900(2) Å, and Z = 4. The final refinement used 877 reflections and gave a residual R value of 0.041. The complex has slightly compressed octahedral coordination, with the zinc atom bound to two monodentate carboxylate groups lying in trans positions and four water molecules. X-ray data and infrared spectra show the Mn, Co, Ni, Zn and Cd complexes to be isostructural with the Zn compound. The electronic, infrared and ESR spectra of the copper(II) complex are consistent with a CuO_4? based chromophore involving two water molecules and two monodentate carboxylate groups in the metal plane, and long axial contacts.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Variations in (Ca + Mg)-ATPase, its inhibitor protein and calmodulin of density (age) separated rabbit erythrocytes

Density (age) separated rabbit erythrocytes were examined for differences in the activities of calmodulin and the protein inhibitor of membrane (Ca²⁺ + Mg²⁺)-ATPase (Lee, K.S. and Au, K.S. (1983) Biochim. Biophys. Acta 742, 54–62) as well as response of the ATPase towards these protein modulators. It was found that activities of the cytosol protein-bound and free inhibitor as well as membrane-bound inhibitor were higher in top (young) cells as compared to bottom (old) cells. Though the activity of the divalent cation associated membrane calmodulin pool was also higher in young cells, calmodulin activity in the erythrosol remained constant in cells from both age groups. The pool of membrane-associated inhibitor was shown to have greater influence on the ATPase than the membrane-associated calmodulin pool. The influence was more pronounced with inhibitor derived from old than from young cell membranes. Response of the young cell ATPase towards the protein inhibitor was better than the old cell enzyme at low inhibitor concentration. At higher inhibitor concentration, however, response of the ATPase from both cell types was similar.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.