Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria
✍ Scribed by Bernard Lardeux; Betty Gouhot; Marguerite Forestier
- Publisher
- Elsevier Science
- Year
- 1983
- Tongue
- English
- Weight
- 414 KB
- Volume
- 131
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
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 5 1.5% 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 P-Nacetylglucosaminidase, 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.