Permeability properties of peroxisomes in digitonin-permeabilized rat hepatocytes. Evidence for free permeability towards a variety of substrates.
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 H2O2 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 H2O2 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-alpha-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.
Verleur
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Department of Clinical Biochemistry, University Hospital Amsterdam, The Netherlands.
Wanders
R J
RJ
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008830:Microbodies / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D014503:Urate Oxidase / Q000378:metabolism
D000429:Alcohol Oxidoreductases; C021380:L-2-hydroxyacid oxidase; D003605:D-Amino-Acid Oxidase; D014503:Urate Oxidase; D005978:Glutathione; D004072:Digitonin