Purification of peroxisomal fraction from rat brain.
A purification procedure to obtain peroxisomes (microperoxisomes) from the brain of suckling rats is reported. A P2 fraction, (crude light mitochondria) frozen and thawed seven times, was subfractionated yielding a P4 fraction, 4-fold enriched in catalase activity with respect to the cytoplasmic extract S1. The P4 fraction was used for further purification of peroxisomes by isopicnic centrifugation on Nycodenz gradient (1.10-1.20 g/ml). When the cerebellum was not included in the starting material, the equilibrium density of peroxisomes was 1.152-1.162 g/ml. In this case the overall yield of catalase in the most enriched fraction was 7% and its relative specific activity more than 50. When the cerebellum was included in the total homogenate, the equilibrium density shifted towards higher values (1.177 g/ml) and in this case the catalase relative specific activity in the peroxisomal enriched fraction was extremely high (> 100). The biochemical results, together with the electron microscope examination of the purified fractions, demonstrate that our procedure allows the best purification of brain peroxisomes so far obtained. The different equilibrium densities of peroxisomes observed in the two sets of experiments are interpreted in terms of size heterogeneity of these organelles in different brain portions and cell types.
Cimini
A M
AM
Department of Science and Biomedical Technology, University of L'Aquila, Italy.
Moreno
S
S
Giorgi
M
M
Serafini
B
B
Cerú
M P
MP
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001933:Brain Stem / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D004798:Enzymes / Q000032:analysis; D005260:Female; D015151:Immunoblotting; D008297:Male; D008830:Microbodies / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D014461:Ultracentrifugation / Q000379:methods
D015415:Biomarkers; D004798:Enzymes; D009419:Nerve Tissue Proteins