Rapid preparation of a distinct lysosomal population from myelinating mouse brain using Percoll gradients.
To study the vesicular lysosome-associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000-17,500-g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self-generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like beta-galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is approximately 100-fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains approximately 12-14% of the total acid hydrolase activities, with a protein yield of approximately 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an approximately 90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.
Caimi
L
L
Department of Biomedical Sciences and Biotechnologies, Medical School, University of Brescia, Italy.
Marchesini
S
S
Aleo
M F
MF
Bresciani
R
R
Monti
E
E
Casella
A
A
Giudici
M L
ML
Preti
A
A
D000818:Animals; D001921:Brain / Q000502:physiology / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D008247:Lysosomes / Q000201:enzymology / Q000648:ultrastructure*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D009186:Myelin Sheath / Q000502:physiology*; D011205:Povidone; D012822:Silicon Dioxide; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure
C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone