Methods for Assay of Ganglioside Catabolic Enzymes

Methods Mol Biol. 2018:1804:383-400. doi: 10.1007/978-1-4939-8552-4_18.

Abstract

Cell plasma membrane gangliosides content and pattern is finely regulated by a complex metabolic machinery. Among different pathways, past and new evidence suggests an important role of the sphingolipid catabolic enzymes associated with both lysosomes and plasma membrane. Over the years, several cell-free and cell-based assays are developed in order to evaluate the activities both intracellularly and at the cell surface. Here, we propose a selection of the most efficient, sensitive, and specific assays that allow determining the activity of the main gangliosides-glycohydrolases such as sialidases, ß-hexosaminidases, ß-galactosidases, and ß-glucosidases in both cell/tissue lysates and directly in living cells.

Keywords: Assays on living cells; Fluorigenic substrates; Glycohydrolases; Lysosome; Plasma membrane; Radioactive substrates; Sialidases.

MeSH terms

  • Animals
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Enzyme Assays / methods*
  • Gangliosides / metabolism*
  • Glucosylceramidase / metabolism
  • Glycoside Hydrolases / metabolism
  • Humans
  • Radioactivity
  • Substrate Specificity
  • beta-Galactosidase / metabolism

Substances

  • Gangliosides
  • Glycoside Hydrolases
  • beta-Galactosidase
  • Glucosylceramidase