Purification and characterization of goat lysosomal beta-mannosidase using monoclonal and polyclonal antibodies

J Biol Chem. 1992 Mar 25;267(9):6178-82.

Abstract

Goat beta-mannosidase was purified 120,000-fold in 26% yield from kidney using concanavalin A-Sepharose chromatography followed by immunoaffinity and cation-exchange chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining, the purified enzyme preparation consists of 90- and 100-kDa peptides. Both these peptides react with anti-beta-mannosidase monoclonal antibodies and produce similar electrophoretic peptide patterns when subjected to limited proteolysis. Deglycosylation reduces the size of the 90- and 100-kDa peptides to 86 and 91 kDa, respectively. Goat kidney tissues lacking beta-mannosidase activity, acquired from animals affected with beta-mannosidosis, do not contain detectable quantities of the 90- and 100-kDa peptides as judged by monoclonal antibody reactivity. We postulate that the 90- and 100-kDa peptides represent two related forms of beta-mannosidase.

Publication types

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

MeSH terms

  • Animals
  • Antibodies
  • Antibodies, Monoclonal
  • Chromatography, Affinity / methods
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation
  • Goats
  • Kidney / enzymology*
  • Kinetics
  • Lysosomes / enzymology*
  • Mannosidases / chemistry
  • Mannosidases / immunology
  • Mannosidases / isolation & purification*
  • Molecular Weight
  • Peptide Mapping
  • beta-Mannosidase

Substances

  • Antibodies
  • Antibodies, Monoclonal
  • Mannosidases
  • beta-Mannosidase