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 (3-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,6-glucuronidase . The ratio of ß-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 .