Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria.
A method for the preparation of lysosomes from rat liver is presented. The procedure requires only standard equipment and is completed within less than 3 h. Homogenization and differential centrifugation were performed at pH 7.4 in isotonic potassium phosphate-buffered sucrose medium. The addition of potassium phosphate, at the concentration used (10 mM), accelerated the sedimentation rate of mitochondria without altering that of lysosomes resulting in the decrease in the mitochondrial contamination of the final pellet. Further purification was achieved by isopycnic centrifugation in 45% isotonic Percoll performed in an angle rotor. Lysosomal fractions representing 51.5% of the original population were recovered over a density range of 1.09 to 1.15 g/ml. The most purified fraction (37-fold purified) contained 25.3% of lysosomal beta-N-acetylglucosaminidase, and only 0.9% of mitochondrial monoamine oxidase and 0.6% of peroxisomal urate oxidase original activities. It was practically devoid to endoplasmic reticulum contamination.
D000118:Acetylglucosaminidase / Q000032:analysis; D000818:Animals; D002021:Buffers; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008099:Liver / Q000648:ultrastructure*; D008247:Lysosomes; D008297:Male; D008930:Mitochondria, Liver; D008995:Monoamine Oxidase / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D002021:Buffers; D008995:Monoamine Oxidase; D000118:Acetylglucosaminidase