Reinvestigation of the sulfhydryl reactivity in bovine brain S100b (.beta..beta.) protein and the microtubule-associated .tau. proteins. Calcium stimulates disulfide cross-linking between S100b .beta.-subunit and the microtubule-associated .tau.(2) protein

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Abstract
Zn2+ and Ca2+ affect the conformation of bovine brain S100b (.beta..beta.) protein and the exposure of its Cys-84.beta., Zn2+ binding to high-affinity sites of native S100b protected the sulfhydryl groups against the thiol-specific reagent 5,5''-dithiobis(2-nitrobenzoate) and antagonized the Ca2+-stimulated reactivity of Cys-84.beta. toward the reagent. Spectroscopic studies on the fluorescence properties of labeled S100b with the fluorescent probes bimane and acrylodan at Cys-84.beta. confirmed the antagonist effect of Ca2+ and Zn2+ with respect to the conformational properties of the protein. Measurements of fluorescence dynamics on bimane-labeled S100b indicated that the slow monomer-dimer equilibrium that characterizes the apoprotein at micromolar concentrations was shifted to the monomer form in the presence of Zn2+, a fact that could explain the previously reported Zn2+-dependent increase of S100b protein affinity for calcium. The difference in the effects of Ca2+ and Zn2+ on the reactivity of Cys-84.beta. in S100b was confirmed when we observed that Ca2+ and Zn2+ have opposite actions on the formation of disulfide bridges between Cys-84.beta. of the S100b .beta.-subunit and sulfhydryl groups on the microtubule-associated .tau.(2) protein. Ca2+ stimulated the covalent complex formation whereas Zn2+ inhibited it. We suggest that Zn2+ may have a modulatory function on Cys-84.beta. reactivity in the S100b .beta.-subunit in vivo. Two types of divalent complexes between .tau.(2) and .beta.-subunit were formed in the presence of Ca2+, an equimolar complex .sigma.(2)-.beta.1 and a complex of one molecule of .tau.(2) with two .beta.-subunits, .tau.(2)-.beta.2. At pH 8.3, the dissociation constant for the equimolar complex was estimated to be approximately 0.3 .mu.M, and the rate constant for the cross-linking reaction was estimated to be 0.2 .mu.M.cntdot.min-1. The rapid attack of .tau.(2) in rupturing disulfide bridges in oxidized S100b demonstrated the strong nucleophilicity of the sulfhydryl group(s) in .sigma.(2). Analogous attacks on other proteins, however, did not lead to disulfide complex formation, indicating that the specific, noncovalent complexation between .tau. protein and S100b preceded disulfide formation and interchange. The result is a rather specific production of disulfide oligomers. The insoluble oligomers of the neurofibrillary tangle that characterizes Alzheimer''s brain might be an example of such a situation.

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