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 p rocedure. 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 molecula r 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, b ut the other stained many bands on immunoblots of whole cell preparati ons. Using fluorescently labelled secondary antibodies we have determi ned that both antisera stain digestive vacuoles in the Acanthamoeba cy toplasm, and do not stain the contractile vacuole. The multi-specific antiserum had high avidity for beta hex, but also stained the carbohyd rate portion of other molecules. These other molecules may be lysosoma l enzymes as well, since the activity of several other lysosomal enzym es was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuo lar structures in Acanthamoeba cytoplasm.