Open AccessPermeability properties of peroxisomes in digitonin‐permeabilized rat hepatocytes
Author(s) -
VERLEUR Nicolette,
WANDERS Ronald J. A.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18353.x
Subject(s) - digitonin , peroxisome , biochemistry , horseradish peroxidase , chemistry , cytosol , catalase , oxidase test , peroxidase , enzyme , biophysics , biology , gene
In order to investigate the permeability properties of rat‐liver peroxisomes in situ , we selectively permeabilized hepatocytes with digitonin in a medium mimicking the cytosol. This system permitted us to study the latency of peroxisomal oxidases by means of measurement of their activities in permeabilized compared to disrupted hepatocytes. The activity of peroxisomal oxidases was studied using three different methods: (1) measurement of the oxidase‐mediated production of H 2 O 2 in a system containing homovanillic acid, horseradish peroxidase and azide; (2) measurement of the rate of substrate utilization or product formation; (3) measurement of the production of H 2 O 2 via the peroxidative action of catalase in the presence of an excess of methanol. The results obtained depended on which system was used to measure the activity of the different oxidases. Our observations lead us to conclude that method 1 cannot be used for latency studies, whereas methods 2 and 3 are suitable under defined circumstances. Based on the results of methods 2 and 3, we conclude that urate oxidase, L‐α‐hydroxyacid oxidase A and D‐amino acid oxidase show no structure‐linked latency in digitonin‐permeabilized hepatocytes, suggesting that the substrates for these enzymes permeate freely through the peroxisomal membrane.