Enzyme inactivation via disulphide-thiol exchange as catalysed by a rat liver membrane protein.
1. The inactivation of cytosol enzymes by a rat liver membrane protein was studied with crude microsomal fraction, plasma membranes or a partially purified preparation of inactivation factor. 2. Complete inactivation of (125)I-labelled glucose 6-phosphate dehydrogenase (EC 1.1.1.49) by membranes did not result in any detectable change in molecular weight when the products were analysed by gradient polyacrylamide-gel electrophoresis. 3. Inactivation of radioactive enzyme was not accompanied by extensive binding to the membrane surface. The maximum extent of binding was 15% of the total enzyme labelled, and bound radioactivity was released only slowly, mainly as trichloroacetic acid-insoluble material. 4. Treatment of membranes with dithiothreitol destroyed the inactivation capacity, whereas the thiol-alkylating agent iodoacetamide had no effect. Partial restoration of the inactivation capacity of reduced membranes after exposure to air was prevented by membrane alkylation with iodoacetamide. 5. Modification of enzyme thiol groups during inactivation was determined by measuring a decrease in iodoacetamide-reactive groups in purified glucose 6-phosphate dehydrogenase. 6. Incubation of membrane-inactivated glucose 6-phosphate dehydrogenase with dithiothreitol resulted in a partial restoration of enzyme activity. 7. As a result of these experiments it is concluded that inactivation proceeds by a disulphide-thiol exchange mechanism. The proposal that this reaction could be involved in the initial step of enzyme degradation is discussed.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003600:Cytosol / Q000201:enzymology; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors; D008099:Liver / Q000032:analysis*; D008565:Membrane Proteins / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000032:analysis; D009249:NADP / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds
D004220:Disulfides; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D004229:Dithiothreitol