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Rat Brain S100b Protein: Purification, Characterization, and Ion Binding Properties. A Comparison with Bovine S100b Protein
Author(s) -
Baudier Jacques,
Labourdette Gérard,
Gerard Dominique
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb07115.x
Subject(s) - chemistry , protein g , calcium , protein a/g , binding protein , biochemistry , gel electrophoresis , microbiology and biotechnology , biology , antibody , fusion protein , recombinant dna , organic chemistry , gene , immunology
We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea‐sodium dodecyl sulfate‐polyacrylamide gel with bovine S100b protein. The properties of the Ca 2+ and Zn 2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal‐free form as well as in the presence of Ca 2+ or Zn 2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCI, rat brain S100b protein is characterized by two high‐affinity Ca 2+ binding sites with a K D of 2 + 10 −5 M and four lower affinity sites with K D about 10 −4 M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low‐affinity calcium binding sites whereas similar Ca 2+ ‐induced conformational changes were observed for both proteins. In the presence of 120 m M KCI rat brain S100b protein bound two Zn 2+ ‐ ions/mol of protein with a K D of 10 −7 M and four other with lower affinity ( K D + 10 −6 M ). The occupancy of the two high‐affinity Zn 2+ binding sites was responsible for most of the Zn 2+ ‐induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn 2+ binding to rat S100b was observed. Such results significantly differ from that obtained with bovine S100b protein, which on binding of eight Zn 2+ equivalents/mol undergoes maximal conformational changes associated with a sixfold increase of the tyrosine fluorescence quantum yield. Finally, we report in this work that zinc binding on rat S100b protein regulates calcium binding by increasing the calcium affinity of the protein and reducing the antagonistic effect of K + on calcium binding.