Calcium-dependent affinity chromatography of S-100 and calmodulin on calmodulin antagonist-coupled Sepharose.

Abstract

Two different calcium-binding proteins, S-100 and calmodulin, have been isolated from bovine brain by calcium-dependent affinity chromatography on calmodulin antagonist coupled to Sepharose. Calmodulin antagonist N-(6-aminohexyl)-5-chloro-l-napthdenesulfonamide (W-7) has been coupled to epoxy-activated Sepharose 6B or cyanogen bromide-acitivated Sepharose 4B. S-100-like protein bound to W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca2+ was eluted with the buffer containing 4 m~ ethylene glycol bis(j3-aminoethyl ether)-N,NJ?’,iV-tetraacetic acid (EGTA) instead of Ca2+. This protein did not stimulate cyclic nucleotide phosphodiesterase and was identified as S-100 by demonstrating its cross-reaction with rabbit antisera to bovine 5-100. Amino acid composition together with electrophoretic evidence shows that S-100-like protein is a mixture of S-100a and S100b. Calmodulin-like protein bound to W-7 coupled to cyanogen bromide-activated Sepharose 4B in the presence of Ca2+ was eluted with buffer containing 4 m~ EGTA. This protein could stimulate cyclic nucleotide phosphodiesterase and had an amino acid compostion and electrophoretic mobility very similar to those of calmodulin, indicating that this protein is calmodulin. When N-6-aminohexyl)-l-naphthalene sulfonamide (W5), a chloro-deficient analogue which has lower affinity for calmodulin than W-7, was coupled to epoxy-activated Sepharose 6B, calmodulin instead of S-100 did bind to this Sepharose in the presence of Ca2+ and was eluted with the buffer containing 4 mM EGTA. These results indicate that different immobilized naphthalenesulfonamide derivatives interact differently with S100 and calmodulin in a calcium-dependent manner and provide a rapid purification procedure for S-100 and calmodulin.