Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.     

Luminescence studies on RbI:Tb3+ crystals.

Optical absorption, photoluminescence, thermoluminescence (TL) and photostimulated luminescence (PSL) studies on RbI:Tb(3+) crystals irradiated with gamma-rays is reported. Photoluminescence of these crystals exhibits characteristic Tb(3+) emissions, due to transitions from the (5)D(3) and (5)D(4) levels to various levels of the (7)F septet. On F-bleaching the gamma-irradiated crystals, Z(3) centres are observed. The TL glow curve indicates a two-step thermal annihilation process for the radiatively created defects. The presence of the characteristic emissions due to terbium ions in the photostimulation at the F-band, and TL emissions under both glow peaks, confirm the participation of Tb ions in the defect production and recombination processes. Trap parameters for the TL process are calculated and presented. The low temperature glow peak is attributable to Z(3) centres.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Further studies on the specific interaction of S-100 protein with synaptosomal particulates.

Abstract— The specific interaction of S-100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca²⁺ -dependent. Two affinities affect the interaction (K_ins= 7.04 × 10^?9 M. 1.28 × 10¹² binding sites/ mg protein; K_ins2= 3.91 × 10^?7M, 2.96 × 10¹³ binding sites/mg protein). The half-saturation time is about 5.5 min at 37°C. The half-life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one-third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S-100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane-bound fraction of S-100.     

Is Tb3+ fluorescence enhancement only due to binding to single stranded polynucleotides.

Enhancement of Tb3+ fluorescence upon binding to double-stranded ribo- and deoxyribo-duplexes was investigated. It was observed that certain double stranded ribopolynucleotides completely quenched the Tb3+ fluorescence and others did not. It is concluded that the nature of the base in the duplex is critical for this enhancement. - Polydeoxyduplexes also showed enhancement of Tb3+ fluorescence, but much higher terbium concentrations were necessary to obtain similar fluorescence signals, indicative of unspecific effects. CD spectra evidence considerable conformational changes of these duplexes, in particular poly(dG-C) . poly(dG-C( which assumes the Z-form in 0.1 nM Tb3+.     

Vanadyl binding to a testicular S-100-like protein and to calmodulin: Electron paramagnetic resonance spectra of VO2+-protein complexes

The binding of vanadyl to a porcine and bovine testicular S-100-like protein and to calmodulin was demonstrated using X-band (9.2 gHz) electron paramagnetic resonance (EPR) spectroscopy in aqueous solution at pH 7.4. In liquid solutions at 22°C, the vanadyl-protein complexes yielded VO²⁺ near rigid limit spectra. At 122 K, each of the three high-field resonances (i.e., 3/2, 5/2, and 7/2 parallel components) splits into two components indicating the presence of two classes of vanadyl-binding sites in each protein. The spectra of the frozen solutions were simulated to give parallel and perpendicular components of the hyperfine coupling constant and g factors similar to other vanadyl-protein complexes.     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Vanadyl binding to a testicular S-100-like protein and to calmodulin: Electron paramagnetic resonance spectra of VO2+-protein complexes

The binding of vanadyl to a porcine and bovine testicular S-100-like protein and to calmodulin was demonstrated using X-band (9.2 gHz) electron paramagnetic resonance (EPR) spectroscopy in aqueous solution at pH 7.4. In liquid solutions at 22°C, the vanadyl-protein complexes yielded VO²⁺ near rigid limit spectra. At 122 K, each of the three high-field resonances (i.e., 3/2, 5/2, and 7/2 parallel components) splits into two components indicating the presence of two classes of vanadyl-binding sites in each protein. The spectra of the frozen solutions were simulated to give parallel and perpendicular components of the hyperfine coupling constant and g factors similar to other vanadyl-protein complexes.     

Spectroscopic studies of metal ion binding to a tryptophan-containing parvalbumin

The binding of Ca(II) and members of the trivalent lanthanide ion, Ln(III), series to apoparvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) results in the development of a distinctive sharp feature in the UV absorption spectrum at about 290 nm. Titration curves obtained by monitoring the spectral change in this region reveal a change in slope after the addition of 1 equiv of metal ion and no further rise after 2 equiv has been added, consistent with sequential binding to the principal EF and CD sites. Laser-induced luminescence excitation spectra of the 7F0----5D0 transition of bound Eu(III) demonstrate the quantitative binding of this ion to the principal sites and disclose the presence of a subsidiary site at pH values greater than 6. Metal ion competition experiments monitored by means of this excitation transition show that the early members of the Ln(III) ion series bind more tightly than those at the end. Tryptophan-sensitized Tb(III) luminescence reveals that this ion binds sequentially to the EF and CD sites, in that order. The intrinsic tryptophan fluorescence of apoparvalbumin is increased in a stepwise fashion as Ca(II) or Ln(III) ions bind sequentially, with the exceptions of Eu(III) and Yb(III). The binding of the latter two ions causes quenching of the protein fluorescence via an energy-transfer process which involves low-lying charge-transfer bands. The distance dependences of the tryptophan to Tb(III) and tryptophan to Eu(III) energy-transfer processes are observed to be identical, consistent with a F?rster-type mechanism in both cases.     

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]₂, and poly-[d(G-C)]₂ has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]₂ suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]₂ than for poly[d(A-T)]₂, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]₂ major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127–143, 1998     

S-100 protein stimulates cellular proliferation

Summary S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [³H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95–31.25 g/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 g/ml). Maximum stimulation of PBL was at 31.25 g/ml. PBL were not inhibited even at high concentrations of S-100p (125 g/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an autocrine tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.     

Studies on the alpha-subunit of bovine brain S-100 protein.

A method is described for the rapid purification of both S-100 protein and calmodulin from crude bovine brain extracts by the use of a fluphenazine-Sepharose affinity column eluted stepwise with decreasing concentrations of free Ca2+. Protein containing only alpha-subunit was purified from preparations of S-100 protein by anion-exchange chromatography. This protein co-migrated with the alpha-subunit of S-100 protein on sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis and had an amino acid composition identical with that previously reported for this subunit. The results of u.v.-absorption and fluorescence-emission spectroscopy indicate that the tryptophan residue of the purified alpha-subunit of S-100 protein undergoes a Ca2+-induced change in environment. Measurements of changes in tryptophan fluorescence with increasing Ca2+ concentrations suggest an apparent dissociation constant of the alpha-subunit for Ca2+ of 7 X 10(-5)M in the absence of K+. In the presence of 90mM-K+ this value is increased to 3.4 X 10(-4)M.     

S-100 protein in the testis

Summary S-100, a protein originally believed to be unique to the nervous system, has recently been found in extraneural cell types. We report here on the presence of S-100 in the testis, namely in Leydig cells and in lymphatic endothelial cells, using immunohistochemical and immunochemical methods. We show that the protein in the testis is immunologically identical to brain S-100. The S-100-labelled cells in the testis exhibit morphological similarities with other cell types in different tissues known to contain S-100.     

Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A.

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.     

A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.     

A subnanosecond-pulse fluorometric study of the CA2+ and MG2+ induced conformational changes on S-100a protein

The fluorescence parameters of the single tryptophan residue (Trp 90 alpha) in S-100a (alpha beta) protein have been studied by steady state fluorimetry and by subnanosecond fluorescence decays excited by pulsed-picosecond laser system. At pHs 7.1 and 8.5, double exponential decays were consistently observed. At both pHs, Ca2+ and to a less extent Mg2+ ions proved to modify the percentage contribution of the two decaying species. The interest of the finding is discussed.