Rapid Preparation of a Distinct Lysosomal Population from Myelinating Mouse Brain Using Percoll Gradients

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Abstract
To study the vesticular lysome-associated transport an the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal populaton starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,00-17,500-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 .apprx. 100-fold as compared with the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosmal preparation contains .apprx. 12-14% of the total acid hydrolase activities, with a protein yield of .apprx. 0.12%. Electron microscopy shows that the lysosomal fraction is composed of an .apprx. 90% pure populaton of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.