Effect of Anions on the Binding and Oxidation of Divalent Manganese and Iron in Modified Bacterial Reaction Centers

The influence of different anions on the binding and oxidation of manganous and ferrous cations was studied in four mutants of bacterial reaction centers that can bind and oxidize these metal ions. Light-minus-dark difference optical and electron paramagnetic resonance spectroscopies were applied to monitor electron transfer from bound divalent metal ions to the photo-oxidized bacteriochlorophyll dimer in the presence of five different anions. At pH 7, bicarbonate was found to be the most effective for both manganese and iron binding, with dissociation constants around 1 μM in three of the mutants. The pH dependence of the dissociation constants for manganese revealed that only bicarbonate and acetate were able to facilitate the binding and oxidation of the metal ion between pH 6 and 8 where the tight binding in their absence could not otherwise be established. The data are consistent with two molecules of bicarbonate or one molecule of acetate binding to the metal binding site. For ferrous ion, the binding and oxidation was facilitated not only by bicarbonate and acetate, but also by citrate. Electron paramagnetic resonance spectra suggest differences in the arrangement of the iron ligands in the presence of the various anions.     

Principle of parametric ion separation in a molecular model of the sodium pump

Expressions were obtained for separation coefficients of sodium and potassium ions in macroscopic and molecular models of the sodium pump. Conjugation between the energy-donor process at ATP hydrolysis and ion transport is achieved at the expense of synchroneous changes of the affinity of ion-binding centers and divided cations. i. e. the cyclic changes of energetic profiles in the membrane for each type of ions. The division coefficient in the stationary state is equal to the product of relative changes in selectivity towards the cations of the enzyme ion-binding centers in phosporylated and unphosphorylated states.     

Propolypeptide and mature portions of von Willebrand factor of bovine origin recognize different sites on type-I collagen obtained from bovine tendon.

We compared the binding of propolypeptide and mature portions of von Willebrand factor of bovine origin to fibrillar type-I collagen obtained from bovine tendon. The propolypeptide (pp-vWF) and the mature portion (m-vWF) of human origin consist of 741 and 2050 amino acids, respectively, and are rather large proteins. The collagen-binding properties of the two proteins of bovine origin were similar in that both bound more avidly to native collagen than to heat-denatured collagen. Bindings was affected similarly by ionic strength but was not modified either by divalent cations or a synthetic peptide containing Arg-Gly-Asp. However, the binding sites in the fibrillar type-I collagen molecule for pp-vWF and m-vWF seem to be different: the two proteins did not effectively compete with each other for binding to collagen. Furthermore, pepsin treatment of fibrillar type-I collagen resulted in a drastic decrease in the binding of pp-vWF, while only a moderate decrease in the binding of m-vWF was observed after the treatment.     

The ordering of corneal collagen fibrils with increasing ionic strength

The fixed stromal charge of bovine corneas, osmotically clamped at physiological hydration, was altered by regulating the amount of chloride ions bound to the matrix. We measured the local fibrillar collagen order using X-ray diffraction methods. As the bound anions increased up to physiological values, the local fibrillar order increased to an optimal value. The coherence distance (t) approximately doubles to a maximum value (409 nm) from 10 mM NaCl to 154 mM NaCl. This then slowly decreased as the bathing solution increased to 1000 mM. In contrast the diameter of the collagen fibrils were minimal at physiological NaCl.     

Effects of lysosomal collagenolytic enzymes, anti-inflammatory drugs and other substances on some properties of insoluble collagen

1. An enzyme system present in a rat liver lysosome-rich fraction was found to liberate soluble hydroxyproline-containing products from insoluble collagen, with maximum activity at pH3·45. It was concluded that a form of cathepsin D was involved since synthetic substrates specific for trypsin were not hydrolysed. Collagenolysis was enhanced by thiol compounds and inhibited by Cu²⁺ ions and the anti-inflammatory drugs phenylbutazone and ibufenac. 2. The possibility that behaviour of collagen and collagenolysis were modified by various substances, either by destruction of intramolecular and intermolecular bonds in tropocollagen or by electrostatic interactions, is discussed. Insoluble collagen was found to bind electrostatically to chondromucoprotein. This interaction was inhibited by some anti-inflammatory drugs. 3. Possible roles of the lysosomal collagenolytic enzyme system in experimental lathyrism in rats given penicillamine, and in erosion of cartilage in rheumatoid arthritis, are considered. 4. Collagenolysis in vivo, which may depend on complex interrelationships between collagen, chondromucoprotein and metal ions, is discussed in relation to possible effects, both harmful and beneficial, of anti-inflammatory drugs used in rheumatoid arthritis.     

Cooperative binding of hexadecyltrimethylammonium chloride and sodium dodecyl sulfate to cytochrome c and the resultant change in protein conformation

The binding of hexadecyltrimethylammonium chloride (HTAC) and sodium dodecyl sulfate (SDS) to cytochrome c was determined by potentiometric titration and the corresponding changes in protein conformation by circular dichroism (CD). The binding isotherms were biphasic; about 20 surfactant cations or anions were bound to cytochrome c in the first phase. Another 30 or so HTA+ ions were bound in the second phase, which was below the critical micelle concentration of the surfactant, but the binding of dodecyl sulfate ions in the second phase increased sharply near the critical micelle concentration. The binding of both surfactants was highly cooperative and was endothermic; the data in the first phase fitted the Hill plot. The corresponding change in the secondary structure of cytochrome c was small; the CD spectra in the ultraviolet region showed a moderate increase in the helicity in HTAC solution and some changes in conformation in SDS solution. However, the CD spectra for the Soret band indicated a marked change in the local conformation around the heme.     

Relationship between ion distribution and membrane potential during a steady state

Equations were derived showing the relationship between the membrane potential and the quantities which influence it under steady state conditions. Essentially, the membrane potential is caused by the valence and concentration of the non-permeating ions. The permeating ions can modify the membrane potential by altering the relative concentration of the non-permeating ions with respect to the concentration of the permeating ions. For muscle, the sodium cations act as the non-permeating ions in the extracellular environment by the maintenance of some type of active metabolic process and large anions act as the non-permeating ions in the intracellular environment. Both of these non-permeating ions contribute about equally to the maintenance of the resting membrane potential. When the active metabolic process for sodium extrusion breaks down or when acids are added, the membrane potential should decrease. Water should enter the cell when the sodium metabolic process is diminished; water should leave the cell when acids are added. When acid is added, it is expected that the cations potassium and sodium will leave the cell with little or no shift of the chloride ions.     

The action of semicarbazide on the aggregation of the tropocollagen macromolecule

1. A difference in conformation was found between the collagen in solutions treated with semicarbazide hydrochloride and those treated with sodium chloride. This difference could be correlated with the difference in extent of aggregation between the fibrils precipitated from these solutions. 2. The action of semicarbazide hydrochloride depended on the pH and temperature of treatment in a complex manner. At constant temperature semicarbazide enhanced aggregation at pH values less than 4·3, but decreased aggregation was observed at pH values greater than 5·0. At pH 4·3 the effect of semicarbazide on aggregation varied with temperature, the tendency to increased aggregation being more pronounced at 34° and 36–37°. Similar increased aggregation tendencies superimposed on an overall decreased aggregation were observed at these temperatures at pH8·9. 3. A specific binding of semicarbazide to the collagen molecule was indicated.     

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes. A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidylcholine from egg yolk has been followed by 31P NMR at 30 degrees C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution (F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts (Cl- < Br- < NO3-). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to L-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

The competition transport of sodium ions and protons in the cytoplasmic access channel of the Na+,K+-ATPase

The changes in capacitance and conductance of lipid bilayer membranes have been studied with adsorbed membrane fragments containing Na⁺,K⁺-ATPase. These changes have been initiated by fast release of protons from a bound form (“caged H⁺”) induced by an UV flash. The changes of the capacitance in the presence of Na⁺,K⁺-ATPase were affected by the frequency of the applied voltage, pH and the concentration of sodium ions. Addition of sodium ions altered the changes of capacitance caused by a pH jump in the medium due to caged H⁺ photolysis, and the magnitude and sign of this effect depended on the initial pH. These results are explained by competitive binding of sodium ions and protons to the ion-binding sites of the Na⁺,K⁺-ATPase at its cytoplasmic side. The pH at which the sign of the sodium ion effect changed allows the evaluation of the pK of the proton binding site, which is about 7.6.     

Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes

The dependence of electrophoretic mobility of multilamellar liposomes composed of egg phosphatidylcholine (PtdCho), dimyristoyl-glycerophosphocholine (Myr2Gro-P-Cho) and dipalmitoyl-glycerophosphocholine (Pam2-Gro-P-Cho) on the concentration of several cations and anions has been measured. Values of surface densities of binding sites and intrinsic binding constants of ions to liposome membranes were determined by processing the results in the framework of Gouy-Stern theory. Sharp reductions in the positive surface potential of Myr2Gro-P-Cho and Pam2Gro-P-Cho liposomes have been detected at the thermotropic transition of the lipids from the gel to liquid-crystalline phase. Similar alterations of liposome surface potential were revealed at the temperature of pretransition, as well as at about 50 degrees C, in the case of Pam2Gro-P-Cho. A model is suggested for ion binding to PtdCho membranes, according to which the ion-binding sites are considered as point defects (vacancies) in the structure of lipid head-groups arranged over a trigonal lattice.     

Effect of ion adsorption on the electrokinetic properties of erythrocytes

The microelectrophoresis technique was used to determine the dependence of human erythrocyte surface potential on the concentration of various cations and anions. The interpretation of the results is based on the Gouy--Chapman--Stern theory. Values of pK, characterizing the binding of ions to the external surface of erythrocytes, as well as numbers of binding sites per unit area were determined. The affinities of ions for the red cell membrane were shown to decrease in the sequence: H+ greater than Ca2+ greater than Sr2+ greater than Mg2+ greater than Ba2+ greater than Li+ greater than Na+ congruent to congruent to K+ congruent to NH4+ and trinitrophenol greater than IO4- greater than CIO4- greater than salicylate congruent to I- greater than greater than SCN- greater than H2PO4- greater than Br- greater than Cl- greater than HPO4(2-). Changes in the ionic strength of the medium resulted in changes in numbers of exposed ion-binding sites. This phenomenon is interpreted in terms of ionic strength-dependent structural transformations of the cell surface coat.     

Binding of an inert, cationic osmium probe to walls of Bacillus subtilis.

X-ray fluorescence spectroscopy and electron microscopy of unstained specimens have been used to study the binding of chloropentaammineosmium(III) chloride to isolated walls of Bacillus subtilis. Native walls bound 0.220 mumol of the osmium probe per mg (dry weight) of walls, whereas walls which were chemically treated to neutralize the available carboxylate groups of the peptidoglycan bound only 0.040 mumol. Teichoic acid-depleted walls bound 0.210 mumol. Thin sections of all wall types showed the osmium probe to be scattered throughout the wall matrix as a small staining deposit. The results support the idea that the metal ion-binding capacity of these walls is mediated by the available carboxylate groups in the wall fabric.     

Sodium and potassium binding by rat liver cell microsomes

The effects of ion concentration, pH, and presence of competing ions on the sodium and potassium binding properties of rat liver cell microsomes were studied. Typical adsorption isotherms were obtained in the concentration dependence studies, with saturation being reached when 1.2 to 1.4 m.eq. cations were retained per gm. of microsome Kjeldahl nitrogen. The retention was shown to be due to a binding to specific sites rather than to a trapping of the cations. The binding showed a sharp pH dependence in the range 6.0 to 7.5. The presence of one cation depressed the binding of the other, indicating that Na⁺ and K⁺ as well as H⁺ ions compete for the same sites. Potassium was bound slightly more strongly than sodium, while hydrogen was bound about 10⁵ times more strongly than either. Calculations show that the binding follows the simple mass law. Similarities between adsorption by microsomes and adsorption by synthetic cation exchange resins are discussed and compared to some of the characteristics of electrolyte behavior in living systems. A possible ion exchange elution, active cation transport mechanism is suggested, involving the preferential elution of Na⁺ out of the cell by H⁺ ions produced by metabolism.     

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.