Human placental neuraminidase. Activation, stabilization and association with beta-galactosidase and its protective protein.
Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37 degrees C and acid pH. Using beta-galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with beta-galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of beta-galactosidase. The formation of multimeric forms of beta-galactosidase is known to require a 32000-Mr 'protective' protein. Monospecific antibodies against this 'protective' protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000-Mr beta-galactosidase protein and against the 32000-Mr 'protective' protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000-Mr 'protective' protein is present both in association with the beta-galactosidase multimer and with the high-density multimeric form together with neuraminidase. Our data further suggest that association of the 32000-Mr 'protective' protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000-Mr 'protective' protein is known to be absent.
D002268:Carboxypeptidases / Q000302:isolation & purification / Q000378:metabolism*; D043402:Cathepsin A; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D003101:Collodion; D004789:Enzyme Activation; D005260:Female; D005696:Galactosidases / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007120:Immunochemistry; D008247:Lysosomes / Q000201:enzymology; D009439:Neuraminidase / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology*; D011485:Protein Binding; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*
D006023:Glycoproteins; D003101:Collodion; D005696:Galactosidases; D009439:Neuraminidase; D001616:beta-Galactosidase; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A