Occurrence in brain lysosomes of a sialidase active on ganglioside.
A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)
Fiorilli
A
A
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
Venerando
B
B
Siniscalco
C
C
Monti
E
E
Bresciani
R
R
Caimi
L
L
Preti
A
A
Tettamanti
G
G
D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004355:Drug Stability; D005615:Freezing; D005732:Gangliosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000201:enzymology; D009439:Neuraminidase / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013570:Synaptic Membranes / Q000201:enzymology
D005732:Gangliosides; C021298:ganglioside, GD1a; D006923:Hymecromone; D011092:Polyethylene Glycols; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D017830:Octoxynol; D009439:Neuraminidase