Characterization, Purification, and SubceUuIar Localization of Bovine Thyroid Sialidases1

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Abstract
Sialidase activities have been studied in bovine thyroid using sialoglycolipids, sialoglycoproteins, sialo-oligosaccharides and fluorogenic 4-methylumbelliferylα-D-N- acetylneuraminate as substrates. No sialidase activity could be detected towards native glycoprotein substrates. From enzme kinetics, effector data and more convincingly from subcellular studies in became clear that in bovine thyroid at least two sialidase activities were present, a sialyllactitol sialidase confined to the lysosomal membrane and a glycolipid sialidase residing in the plasma membrane and displaying the features of a true ectoenzyme. The lipid requirement for full enzyme activity supported the membrane bound character of both sialidase activities. A soluble sialidase activity could not be demonstrated. After solubilization by CHAPS treatment, partial purification of the sialyllactitol sialidase could be achieved by affinity chromatography (Sepharose diamino dipropylamino-N-acetyleuraminic acid). The purified enzyme was extremely labile. Titration of the sialidase preparation with amino acid modifying gents revealed that sulfhydryl- and typtophanyl groups were essential for the silaidase action.

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