Systematic purification of five glycosidases from Streptococcus (Diplococcus) pneumoniae

Abstract

Five of the six known glycosidases in the culture medium of Streptococcus pneumoniae have been purified from 600to 55,000-fold by a systematic procedure of ion exchange and affinity chromatography. Following partial separation of the glycosidases on DEAE-Sephadex, the neuraminidase, the endo-cu-N-acetylgalactosaminidase, the /3-galactosidase, and the P-iV-acetylglucosaminidase were further purified on agarose affinity adsorbents with ligands derived, respectively, from ovine submaxillary mucin glycopeptides, antifreeze glycoprotein, p-aminophenyl-1-thio-p-o-galactoside, or p-aminophenyl-1-thio-/?-o-N-acetylglucosaminide. The purified enzymes had specific activities from 25 to 48 pmoll minlmg. The endo-P-N-acetylglucosaminidase was also purified further by gel filtration and ion exchange chromatography and a persistent contaminant of P-N-acetylglucosaminidase was removed by adsorption on p-aminophenyl-lthio-P-u-N-acetylglucosaminide-agarose. Each glycosidase preparation was substantially free of contaminating glycosidic, hemolytic, and proteolytic activities. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single polypeptide species for the P-galactosidase, the endo-cu-Nacetylgalactosaminidase, and for the @V-acetylglucosaminidase, corresponding to apparent molecular weights of 350,000, 190,000, and 180,000, respectively. Rapid assay procedures for three of the glycosidases were developed. Substrates for neuraminidase and endo-/3-galactosidase were synthesized by treatment of asialo-q-acid glycoprotein with specific glycosyltransferases to produce either [‘“C]NeuAccu2 + 6Ga1, or [W]GalNAccYl + 3 (Fuccul --f 2)Gal at the nonreducing termini of the oligosaccharide chains. The only ovalbumin glycopeptide, (Asn(GlcNAc),(Man),), that served as a substrate for the S. pneumoniae endo-/3-N-acetylglucosaminidase was labeled in the terminal mannose residues by reduction with NaB3H, after mild periodate treatment.