A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis.
A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and beta-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to beta-glucuronidase. The ratio of beta-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5-8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.
D000135:Acid Phosphatase / Q000032:analysis; D002021:Buffers; D002374:Catalase / Q000032:analysis; D002458:Cell Fractionation; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000201:enzymology; D003576:Electron Transport Complex IV / Q000032:analysis; D004586:Electrophoresis; D005952:Glucose-6-Phosphatase / Q000032:analysis; D005966:Glucuronidase / Q000032:analysis; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008247:Lysosomes / Q000032:analysis / Q000201:enzymology*; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D008861:Microsomes; D008928:Mitochondria / Q000201:enzymology; D008995:Monoamine Oxidase / Q000032:analysis; D011506:Proteins / Q000032:analysis; D013429:Sulfatases / Q000032:analysis; D014020:Tissue Extracts
D002021:Buffers; D011506:Proteins; D014020:Tissue Extracts; D002374:Catalase; D008995:Monoamine Oxidase; D003576:Electron Transport Complex IV; D006867:Hydrolases; D000135:Acid Phosphatase; D005952:Glucose-6-Phosphatase; D013429:Sulfatases; D005966:Glucuronidase