Dual subcellular localization of sialidase in cultured granule cells differentiated in culture

Marina Pitto, Antonella Giglioni, Guido Tettamanti

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Abstract

A rapid small-scale procedure was set up to obtain highly purified preparations of lysosomes and plasma membranes from the homogenate of cerebellar granule cells differentiated in culture. It consisted in a centrifugation of the postnuclear fraction P2, on a Percoll gradient with formation of an upper and lower band. The upper band, upon centrifugation on 1 M sucrose, produced a light band lying on the top, that constituted the plasma membrane preparation. The upper band constituted the lysosome preparation. The plasma membrane preparation exhibited a 6-fold relative specific activity increase of Na+, K+-ATPase and 5′-nucleotidase, with negligible contamination by other subcellular markers; the lysosomal preparation exhibited a 30-fold relative specific activity increase of β-galactosidase and β-hexosaminidase, with virtually no contamination by other subcellular markers. Both the lysosome and plasma membrane preparations carried sialidase activity on MUB-NeuNAc and ganglioside GD1a. The sialidase activity on GD1a required the presence of Triton X-100 in both subcellular preparations; the sialidase activity on MUB-NeuNAc was markedly activated by albumin only in the lysosomes. The lysosomal sialidase had a unique optimal pH value, 3.9. The plasma membrane sialidase featured two values of optimal pH, one at 3.9, for both substrates and second at 5.4 and 6.0 for MUB-NeuNAc and GD1a, respectively. It is concluded that cerebellar granule cells differentiated in vitro possess one lysosomal sialidase and two plasma membrane sialidases, all of them active on ganglioside.

Original languageEnglish
Pages (from-to)367-374
Number of pages8
JournalNeurochemistry International
Volume21
Issue number3
DOIs
Publication statusPublished - 1992

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Neuraminidase
Cultured Cells
Cell Membrane
Lysosomes
Centrifugation
Galactosidases
Hexosaminidases
5'-Nucleotidase
Gangliosides
Octoxynol
Sucrose
Albumins

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Dual subcellular localization of sialidase in cultured granule cells differentiated in culture. / Pitto, Marina; Giglioni, Antonella; Tettamanti, Guido.

In: Neurochemistry International, Vol. 21, No. 3, 1992, p. 367-374.

Research output: Contribution to journalArticle

Pitto, Marina ; Giglioni, Antonella ; Tettamanti, Guido. / Dual subcellular localization of sialidase in cultured granule cells differentiated in culture. In: Neurochemistry International. 1992 ; Vol. 21, No. 3. pp. 367-374.
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