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Rapid Preparation of a Distinct Lysosomal Population from Myelinating Mouse Brain Using Percoll Gradients
Author(s) -
Caimi L.,
Marchesini S.,
Aleo M. F.,
Bresciani R.,
Monti E.,
Casella A.,
Giudici M. L.,
Preti A.
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb07250.x
Subject(s) - percoll , acid phosphatase , lysosome , population , differential centrifugation , organelle , biochemistry , golgi apparatus , biology , hydrolase , centrifugation , isopycnic , enzyme , chemistry , endoplasmic reticulum , demography , sociology
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 (P 2 ), followed by isopycnic centrifugation of fraction P 2 on a self‐generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like β‐galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is ∼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 ∼12–14% of the total acid hydrolase activities, with a protein yield of ∼0.12%. Electron microscopy shows that the lysosomal fraction is composed of an ∼90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.