Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases

Carbohydr Res. 1980 Mar 15;81(2):315-22. doi: 10.1016/s0008-6215(00)85662-1.

Abstract

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylgalactosamine / isolation & purification
  • Acetylgalactosamine / metabolism
  • Clostridium perfringens / enzymology*
  • Galactosidases / metabolism*
  • Hexosaminidases / isolation & purification
  • Hexosaminidases / metabolism*
  • Hydrogen-Ion Concentration
  • Indicators and Reagents
  • Kinetics
  • Spectrophotometry / methods
  • Substrate Specificity
  • alpha-N-Acetylgalactosaminidase
  • beta-Galactosidase / metabolism*

Substances

  • Indicators and Reagents
  • Galactosidases
  • Hexosaminidases
  • beta-Galactosidase
  • alpha-N-Acetylgalactosaminidase
  • Acetylgalactosamine