Purification and characterization of goat lysosomal beta-mannosidase using monoclonal and polyclonal antibodies.
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.
Sopher
B L
BL
Department of Pathology, Michigan State University, East Lansing 48824.
Traviss
C E
CE
Cavanagh
K T
KT
Jones
M Z
MZ
Friderici
K H
KH
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006041:Goats; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D010449:Peptide Mapping; D044902:beta-Mannosidase
D000906:Antibodies; D000911:Antibodies, Monoclonal; D008361:Mannosidases; D044902:beta-Mannosidase