Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 · 10⁴. On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 ·10⁵) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 · 10⁵, and a ganglioside/ protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dinier of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides G_D1a and G_D1b, which, like G_T1b, are potential substrates for the enzyme and G_M1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. G_T1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with G_D1a and G_D1b.