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.     

Coordinative binding of divalent cations with ligands related to bacterial spores. Equilibrium studies

It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0°C; μ = 1.0 KNO₃) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), α,ε-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.     

Anion influence on the binding of divalent cations to phosphatidylcholine

We have used the osmotic pressure technique of Rand, Parsegian and co-workers (Nature 259 (1976) 601–603) to investigate the effect of anion species on the binding of M²⁺ to dipalmitoylphosphatidylcholine bilayers. Calcium and magnesium salts show a complex behavior which is consistent with both anion binding and screening. We observe virtually no change, within the accuracy of our experiment, in the decay of repulsive pressure with inter-bilayer separation for the acetate and nitrate salts of magnesium and calcium; however, the chloride salt does show a different pressure decay. At any given bilayer separation, , with calcium and magnesium salts present, the anions produce a decrease in the repulsive pressure in the order acetate⁻ > Cl⁻ > NO₃⁻.     

Weak binding of divalent cations to plasma gelsolin

Calcium binding of swine plasma gelsolin was examined. When applied to ion-exchange chromatography, its elution volume was drastically altered depending on the free Ca2+ concentration of the medium. The presence of two classes of Ca2+ binding sites, high-affinity sites (Kd = 7 microM) and low-affinity sites (Kd = 1 mM), was suggested from the concentration dependence of the elution volume. The tight binding sites were specific for Ca2+. The weakly bound Ca2+ could be replaced by Mg2+ once the tight binding sites were occupied with Ca2+. The binding of metal ions was totally reversible. Circular dichroism measurement of plasma gelsolin indicated that most change in secondary structure was associated with Ca2+ binding to the high-affinity sites. Binding of Mg2+ to the low-affinity sites caused a secondary structural change different from that caused by Ca2+ bound to the high-affinity sites. Gel permeation chromatography exhibited a small change in Stokes radius with and without Ca2+. Microheterogeneity revealed by isoelectric focusing did not relate to the presence of two classes of Ca2+ binding sites. These results indicated that plasma gelsolin drastically altered its surface charge property due to binding of Ca2+ or Ca2+, Mg2+ with a concomitant conformational change.     

Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.     

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.     

Selective binding of divalent cations toward heme proteins

Potential toxicity of transition metals like Hg, Cu and Cd are well known and their affinity toward proteins is of great concern. This work explores the selective nature of interactions of Cu²⁺, Hg²⁺ and Cd²⁺ with the heme proteins leghemoglobin, myoglobin and cytochrome C. The binding profiles were analyzed using absorbance spectrum and steady-state fluorescence spectroscopy. Thermodynamic parameters like enthalpy, entropy and free energy changes were derived by isothermal calorimetry and consequent binding parameters were compared for these heme proteins. Free energy (DG) values revealed Cu²⁺ binding toward myoglobin and leghemoglobin to be specific and facile in contrast to weak binding for Hg²⁺ or Cd²⁺. Time correlated single photon counting indicated significant alteration in excited state lifetimes for metal complexed myoglobin and leghemoglobin suggesting bimolecular collisions to be involved. Interestingly, none of these cations showed significant affinity for cytochrome c pointing that, presence of conserved sequences or heme group is not the only criteria for cation binding toward heme proteins, but the microenvironment of the residues or a specific folding pattern may be responsible for these differential conjugation profile. Binding of these cations may modulate the conformation and functions of these biologically important proteins.     

Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.     

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.     

Further studies on the formation of cardiolipin and phosphatidylglycerol in rat liver mitochondria. Effect of divalent cations and the fatty acid composition of CDP-diglyceride.

The divalent cation requirement for mitochondrial cardiolipin biosynthesis has been further investigated. The relative order of divalent cation activity was Co-2+ greater than Mn-2+ greater than Mg-2+. Cardiolipin was not formed in the incubations with Zn-2+, Fe-2+, Cu-2+, Hg-2+, and Ca-2+. Cardiolipin synthesis in the presence of optimal cincentration of Co-2+ was inhibited by Ca-2+. A series of CDP-diglycerides was synthesized having differences in fatty acid chain lenth and degree of unsaturation. These compounds were tested in mitochondrial cardiolipin and phosphatidylglycerol synthesis. Although there were some minor differences between phosphatidylglycerol and cardiolipin synthesis, in general, saturated shorter chain CDP-diglycerides (dilauroyl and dimyristoyl) were better substrates than the longer chain dipalmitoyl and distearoyl homologues. Introduction of double bonds into distearoyl CDP-diglyceride resulted in more rapid rates of synthesis (e.g. dioleoyl and dilinoleoyl CDP-diglyceride). Significance of the results is dicussed with regard to possible mechanisms of linoleic acid incorporation into rat liver cardiolipin.     

Potassium-39 and sodium-23 NMR studies of cation binding to phosvitin

The binding of Na+ and K+ to hen egg-yolk phosvitin has been studied by 23Na and 39K NMR. A transition in the binding behaviour of these cations is shown to accompany deprotonation of the phosphate groups on this highly charged protein. At lower pH the data are well described by a purely mass-action binding model, whereas at higher pH significant polyelectrolyte effects are apparent. From single ion as well as competition experiments K+ is shown to bind more strongly to phosvitin than does Na+. The temperature and magnetic field strength dependences of the 23Na and 39K relaxation rates provide a picture of phosvitin as a highly flexible macromolecule. This work demonstrates the potential of 39K NMR as a useful tool in the study of protein-cation interactions.     

Chemical modification of low-density lipoprotein enhances the number of binding sites for divalent cations.

The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations.