Author:
Cramer Jana,Effelsberg Daniel,Girzalsky Wolfgang,Erdmann Ralf
Abstract
This protocol describes the isolation of peroxisomes from Saccharomyces cerevisiae by density gradient centrifugation using a sucrose, OptiPrep, or OptiPrep/sucrose gradient. Oleic acid–induced cells are first converted to spheroplasts using lyticase for cell wall digestion. Spheroplasts are homogenized, and nuclei and cell debris are removed by low-speed centrifugation to produce a postnuclear supernatant (PNS). Separation of the PNS by density gradient centrifugation is suitable for many analytical applications; however, to increase the yield of peroxisomes, further fractionation of the PNS is possible. Differential centrifugation of the PNS allows removal of the cytosol and other contaminating organelles, resulting in an organellar pellet (OP) enriched in peroxisomes and mitochondria that can be loaded onto the density gradient. Following density gradient centrifugation of the PNS or OP, fractions are collected from the bottom of the centrifuge tube. The distribution of organelles, including peroxisome peak fractions, is characterized by measurement of marker enzyme activity.
Publisher
Cold Spring Harbor Laboratory
Subject
General Biochemistry, Genetics and Molecular Biology
Reference10 articles.
1. Heterogeneity of erythrocyte catalase II. Isolation and characterization of normal and variant erythrocyte catalase and their subunits;Eur J Biochem,1974
2. Bergmeyer HU , Gawehn K , Grassi M . 1974. Enzymes as biochemical reagents. In Methods of enzymatic analysis (ed. Bergmeyer HU ), pp. 425–522. Academic, London.
3. Identification of a yeast peroxisomal member of the family of AMP-binding proteins;Eur J Biochem,1996
4. Isolation and primary structure of the gene encoding fructose-1,6-bisphosphatase fromSaccharomyces cerevisiae
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