Isolation of lysosomes from brain tissue. A separation method by means of alteration of mitochondrial and synaptosomal sedimentation properties.
1. A crude mitochondrial-lysosomal preparation from brain tissue was layered on a sucrose gradient containing 20mm-succinate, 10mm-Tris and 1mm-disodium EDTA at pH7.4. The lysosomes were separated from the mitochondria and synaptosomes by means of a twosteps centrifugation procedure. In a first low-speed step (40min at 5300g at 15 degrees C) the sedimentation rate of mitochondria and mitochondria-containing synaptosomes was enlarged due to passage of these subcellular structures through the sucrose gradient with the above-mentioned chemicals (called ;chemical field'). That part of the gradient which contained the mitochondria and synaptosomes was removed and substituted by a gradient suitable for isopycnic isolation of lysosomes in a second centrifugation step. The achieved purification for bovine brain lysosomes was 5-8-fold, for rat brain lysosomes 7-10-fold, over the homogenate. 2. The enlargement of the sedimentation rate of mitochondria and synaptosomes was brought about by the presence of succinate, but also by one of the following salts in the chemical field: malonate, fumarate, pyruvate, phosphate and chloride. 3. Comparison of the chemical-field method with other methods for the isolation of lysosomes showed that (a) with the chemical-field method a 2-3-times higher purification of the rat and bovine brain lysosomal fraction can be achieved than with the procedure described by Koenig, Gaines, McDonald, Gray & Scott [(1964) J. Neurochem.11, 729-743], and that (b) similar purification results for rat liver lysosomes were obtained when the chemical-field method and the procedure described by van Dijk, Roholl, Reijngoud & Tager [(1976) FEBS Lett.62, 177-181] were compared.
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure*; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D008099:Liver; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008722:Methods; D008854:Microscopy, Electron; D008928:Mitochondria / Q000648:ultrastructure; D051381:Rats; D013574:Synaptosomes / Q000648:ultrastructure