Isolation and characterization of peroxisomes from the liver of normal untreated rats

Abstract

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-513] for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals. The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondrial fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12-1.26 g/cm3) and subjected to an integrated force of 1.252 X 10(6) X (g X min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid beta-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.