Purification, characterization, and immunochemical studies of beta-N-acetyl-D-hexosaminidase from the parasitic nematode Trichinella spiralis.
The exoglycosidase, beta-N-acetyl-D-hexosaminidase was purified 600-fold from the muscle-stage larvae (L1) of Trichinella spiralis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme-active fraction contained 4 polypeptides with apparent molecular weights of 100,000, 68,000, 58,000 and 54,000. The beta-N-acetyl-D-hexosaminidase corresponds to the Mr 100,000 polypeptide as demonstrated by SDS-PAGE analysis of the enzyme-stained region isolated from a non-denaturing polyacrylamide gel. In addition, rabbit antiserum to a homogeneous preparation of the Mr 100,000 polypeptide (isolated by electroelution from an SDS-PAGE gel) specifically immunoprecipitated beta-N-acetyl-D-hexosaminidase activity from an extract of L1. Isoelectrofocusing (pH 3-10) resolved 4 isoenzymes of T. spiralis beta-N-acetyl-D-hexosaminidase with isoelectric points (pI) of 5.35, 5.49, 5.63 and 5.79. The T. spiralis beta-N-acetyl-D-hexosaminidase is a glycoprotein based on its binding to lentil-lectin Sepharose affinity column and its specific binding of concanavalin A on Western blots. The IgG fraction of T. spiralis-infected mouse serum specifically immunoprecipitated T. spiralis beta-N-acetyl-D-hexosaminidase. The removal of carbohydrate from T. spiralis beta-N-acetyl-D-hexosaminidase significantly reduced its antigenicity. Immunocytochemical analysis of L1 tissue sections with polyclonal rabbit antisera to the homogeneous beta-N-acetyl-D-hexosaminidase enzyme indicated localization on cell membranes and the epicuticle.
Rhoads
M L
ML
Helminthic Diseases Laboratory, U.S. Department of Agriculture, Beltsville, MD 20705.
D000818:Animals; D000947:Antigens, Helminth / Q000276:immunology; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology; D007525:Isoelectric Focusing; D007814:Larva / Q000302:isolation & purification; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D014234:Trichinella / Q000201:enzymology* / Q000276:immunology / Q000302:isolation & purification; D001619:beta-N-Acetylhexosaminidases / Q000302:isolation & purification* / Q000378:metabolism
D000947:Antigens, Helminth; D007074:Immunoglobulin G; D003208:Concanavalin A; D006026:Glycoside Hydrolases; D001619:beta-N-Acetylhexosaminidases