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The single cysteine on the alpha-subunit of bovine brain S-100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 +/- 2 nm, suggesting that the probe is in a fairly hydrophobic environment. Addition of Ca2+ to the protein caused the emission maximum to undergo a red shift to 504 +/- 2 nm, implying that the fluorophore is now more exposed to the solvent. Zn2+, when added to the protein, induced only a small perturbation and the emission maximum shifted to 481 +/- 2 nm. Ca2+ was able to perturb the fluorophore in the presence of Zn2+. 2-p-Toluidinylnaphthalene-6-sulfonate (TNS)-labelled alpha-subunit when excited at 345 nm exhibited very little fluorescence in the absence of Ca2+. Addition of Ca2+ resulted in an increase in TNS fluorescence accompanied by a blue shift of the emission maximum to 445 +/- 1 nm indicating that the probe in the presence of Ca2+ moves to a hydrophobic domain. The fact that Ca2+ and Zn2+ can perturb the labelled sulfhydryl group in the presence of each other clearly demonstrates that the binding sites for the two metal ions must be different on the alpha-subunit as well as on the S-100a protein.

The size and shape of S-100a and S-100b proteins in solution have been examined by gel filtration and ultracentrifugation in the presence and absence of Ca2+. S-100a and S-100b proteins, in the absence of Ca2+, have an intrinsic sedimentation coefficient, so20,w of 2.20 and 2.15 S, respectively and in 1 mM Ca2+ their so20,w values were decreased to 2.05 and 1.95 S, respectively, indicating an unfolding of the protein molecules. The Stokes radii of S-100a and S-100b (-Ca2+) were 23.4 A and 24.0 A and they decreased to 22.2 A and 22.3 A in the presence of Ca2+. The Ca2+ effect on S-100b greater than S-100a was in agreement with our earlier CD observations. Among the monovalent cations tested (K+, Na+ and Li+) K+ had the maximum effect on the Stokes radii and so20,w values of S-100 proteins. Since certain functions of the nervous system are accompanied by local changes in ionic concentrations of Ca2+, Na+ and K+, it is conceivable that these respective conformational changes induced in S-100 proteins by these metals may be related to their function in the brain.

S-100 protein was examined for its ability to affect microtubule assembly—disassembly in vitro. Addition of S-100 protein inhibited microtubule assembly in a dose-dependent manner in the presence of Ca 2+ , but this effect was not observed in the presence of 2 mM EGTA. The interaction of microtubule proteins with S-100 protein was also demonstrated by Ca + -dependent affinity chromatography on S-100 protein—Sepharose. The proteins bound to the Sepharose column comigrated with tubulin as judged by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. These results might suggest a possible physiological role of S-100 protein in the microtubule assembly—disassembly system.

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.

S-100 protein inhibits the assembly of brain microtubule proteins in vitro in the presence of 10 μM free Ca2+. The S-100 effect is generally greater on the rate than on the extent of assembly, and even greater as the microtubule protein concentration decreases and the time of preincubation between S-100 and microtubule proteins before GTP addition increases, at a given S-100/tubulin dimer molar ratio. The S-100 effect is greatly enhanced in the presence of physiological concentrations of K+ and is completely reversed by EGTA.

The effect of Zn2+ binding on the circular dichroism (CD) spectra of brain-specific S-100a and S-100b calcium-binding proteins has been examined. In the presence of Zn2+, S-100a undergoes a conformational change and the decrease in ellipticity at 222 nm, as a result of Zn2+ addition, was nearly 1400 deg.cm−2.dmol−1, whereas with S-100b there was no significant conformational change. Ca2+ was able to bind to S-100 proteins in the presence of Zn2+ and the two metal-ion binding sites on the proteins appear to be different. In the presence of Zn2+, K+ had not significant effect on the conformation of S-100 proteins. Ca2+ and Zn2+ binding induce different environments around the tyrosine residues in S-100a, whereas with S-100b, similar changes were observed for the single tyrosine residue, using either metal.

Two calcium-binding sites of the Mtsl protein, a member of S-100 protein family, were distinguished with the Fluo-3 fluorescent technique. The geometric mean of the apparent dissociation constant (K^) for these two sites is 2.6 (iM; the Hill coefficient («H) is 0.98. In the presence of a novel target protein p37, isolated from the mouse adenocarcinoma cell line CSML-100, Mtsl binds Ca 2+ ions with higher affinity and with strong positive cooperativity (Ka = 0.2 jiM, «H = 1.91). Interaction of Mtsl with p37 is confirmed by the fluorescent probe 2-/>-toluidinylnaphthalene-6-sulfonate (TNS). Reaction with TNS shows that p37 interacts with the hydrophobic site of Mtsl which is exposed due to the binding of Ca 2+ ions. © 1997 Federation of European Biochemical Societies. Keywords: S-100 protein; Calcium; Mtsl; Fluo-3; Target protein 1. Introduction Mtsl protein belong s to the family of S-100 proteins. This family consists of small Ca 2+ -binding proteins which are ex-pressed in different cell types and tissues [1]. Their biological function is still unclear. Studies on the expression of the