Isolation of Highly Purified Lysosomes from Rat Liver: Identification of Electron Carrier Components on Lysosomal Membranes

Abstract

Using Percoll density gradient centrifugation after treatment of the postnuclear supernatant (PNS) with 1 mM Ca²⁺ to swell and lighten mitochondria, we isolated highly purified lysosomes (dextranosomes) in high yield (25%) from the livers of rats to which dextran had been administered. The lysosomal fraction obtained by this method was enriched more than 100-fold in N-acetyl-β-glucosaminidase and arylsulfatase and 40-fold in acid phosphatase and β-glucosidase. Electron microscopic examination and measurement of marker enzyme activity for various subcellular organella indicated that the lysosomal fraction was essentially free from contamination by other organella. Flavins, ubiquinones, and hemochromes were found on lysosomal membranes and investigated. The FAD and ubiquinone-9 contents of the purified lysosomal membranes were 0.118 and 6.93 nmol/mg of protein, respectively. Hemochromes in lysosomes showed spectra similar to that of a b-type cytochrome, with the α-peak at 562 nm and the γ-peak at 436 nm.