tailieunhanh - Báo cáo Y học: Calcium-binding by p26olf, an S100-like protein in the frog olfactory epithelium

Frog p26olf is a novel S100-like Ca21-binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially, and has a total of four Ca21-binding sites (known as EF-hands). In this study, to elucidate the mechanism of Ca21-binding to each EF-hand (named EF-A, -B, -C and -D from the N-terminus of p26olf), we examined Ca21-binding in wild-type p26olf and also in its mutants in which a glutamate at the –z coordinate position within each Ca21-binding loop was substituted for a glutamine. Flow dialysis experiments showed that the wild-type binds nearly four Ca21 per molecule maximally, while all the. | Eur. J. Biochem. 268 6029-6036 2001 FEBS 2001 Calcium-binding by p26olf an S100-like protein in the frog olfactory epithelium Naofumi Miwa Yukiko Shinmyo and Satoru Kawamura From the Department of Biology Graduate School of Science Osaka University Japan Frog p26olf is a novel S100-like Ca21 -binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially and has a total of four Ca21-binding sites known as EF-hands . In this study to elucidate the mechanism of Ca21-binding to each EF-hand named EF-A -B -C and -D from the N-terminus of p26olf we examined Ca21-binding in wild-type p26olf and also in its mutants in which a glutamate at the -z coordinate position within each Ca21-binding loop was substituted for a glutamine. Flow dialysis experiments showed that the wild-type binds nearly four Ca21 per molecule maximally while all the mutants bind approximately three Ca21. Although EF-B and -D are p26olf-specific EF-hands and their role in Ca21-binding is not known the result unequivocally showed that they actually bind Ca21. The overall Ca21-binding affinity decreased in the three mutants. The decrease was very large in the mutants of EF-A and -B which suggested that the Ca21-affinities are high in EF-A and -B in the wildtype. Assuming the presence of four steps of Ca21-binding we determined the dissociation constant of each step in wild-type p26olf. To assign which step takes place at which EF-hand we measured the antagonistic effect of K1 on each step as the effect of K1 is thought to be a function of the number of the carboxyl groups in an EF-hand. Although the actual Ca21-binding mechanism may not be so simple this study together with the mutation study suggested a tentative Ca21-binding model of p26olf the order of Ca21-binding to p26olf is EF-B EF-A EF-C and EF-D. Based on these results we speculate that similar Ca21-binding takes place in an S100 dimer. Keywords calcium-binding p26olf S100. We have previously isolated a novel