Dual subcellular localization of sialidase in cultured granule cells differentiated in culture.
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 beta-galactosidase and beta-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.
Pitto
M
M
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
Giglioni
A
A
Tettamanti
G
G
D000818:Animals; D002454:Cell Differentiation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D009439:Neuraminidase / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D007527:Isoenzymes; D009439:Neuraminidase