Isolation of N-acetyl-beta-hexosaminidase from Acanthamoeba castellanii.
The lysosomal enzyme N-acetyl-beta-hexosaminidase (beta hex) has been purified from Acanthamoeba castellanii growth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified beta hex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified beta hex was enzymatically deglycosylated and injected into two rabbits to make polyclonal antibodies. One antiserum was specific for beta hex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in the Acanthamoeba cytoplasm, and do not stain the contractile vacuole. The multi-specific antiserum had high avidity for beta hex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures in Acanthamoeba cytoplasm.
D000048:Acanthamoeba / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology / Q000648:ultrastructure; D008970:Molecular Weight; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*
D003470:Culture Media; D001619:beta-N-Acetylhexosaminidases