Improved isolation and purification of rat liver peroxisomes by combined rate zonal and equilibrium density centrifugation.
Two different peroxisome preparations were isolated from male rat liver by using total homogenate (TH) as the starting material for one and the light mitochondrial (L) fraction for the other. The technique worked out is based on rate zonal (RZ) centrifugation in a sucrose gradient and subsequent isopycnic centrifugation in a Nycodenz gradient. The peroxisome fraction isolated from the L fraction consisted of 97-98% peroxisomal protein with catalase activity 49-fold enriched over TH. The peroxisome preparation isolated directly from TH represented about 55% of the total liver peroxisome population and had catalase activity 43-fold enriched compared with TH. The contribution of peroxisome protein to the liver protein was calculated to be in the range 1.82-2.02%. Peroxisomes isolated from TH were considerably more heterogeneous in size than peroxisomes isolated from the L fraction. Comparison of the polypeptide patterns of both preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed some quantitative differences. Several major polypeptides were found to be exclusively located in the peroxisome membrane. These polypeptides migrated in the gel with apparent molecular masses of 69, 42.5, 36, 26, 21, and 15 kDa.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002501:Centrifugation, Zonal; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008830:Microbodies / Q000201:enzymology / Q000648:ultrastructure*; D008930:Mitochondria, Liver / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains