Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol.
Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% (w/w) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and beta-D-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Miguel Servet, Zaragoza, Spain.
J
J
Osada
H
H
Aylagas
J
J
Sánchez-Prieto
I
I
Sánchez-Vegazo
E
E
Palacios-Alaiz
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D003911:Dextrans; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000648:ultrastructure*; D008247:Lysosomes / Q000032:analysis* / Q000201:enzymology; D008297:Male; D008722:Methods; D008930:Mitochondria, Liver / Q000032:analysis; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D001616:beta-Galactosidase / Q000378:metabolism
D003911:Dextrans; D008055:Lipids; D011092:Polyethylene Glycols; D000135:Acid Phosphatase; D001616:beta-Galactosidase