Purification and partial characterization of lysosomal neuraminidase from human placenta

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Abstract
Lysosomal neuraminidase and β‐galactosidase are present in a complex together with a 32‐kDa protective protein. This complex has been purified and the different components have been dissociated using potassium isothiocyanate (KSCN) treatment. β‐Galactosidase remains catalytically active, but neuraminidase loses its activity upon dissociation. The inactive dissociated neuraminidase was purified by removing the remaining non‐dissociated β‐galactosidase/protective protein complex using β‐galactosidase‐specific affinity chromatography. The dissociated neuraminidase material shows two major polypeptides on SDS‐PAGE with an apparent molecular mass of 76 kDa and 66 kDa. Subsequently the 32‐kDa protective protein was dissociated from the β‐galactosidase/protective protein complex, and purified.Antibodies raised against the dissociated inactive neuraminidase preparation specifically immunoprecipitate the active neuraminidase present in the complex with β‐galactosidase and protective protein.By immunoblotting evidence is provided that the 76‐kDa protein is a subunit of neuraminidase which, in association with the 32‐kDa protective protein, is essential for neuraminidase activity.