Enzyme inactivation via disulphide–thiol exchange as catalysed by a rat liver membrane protein

Abstract

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.

Effect of physiological reducing compounds on the inactivation of tyrosine aminotransferase from guinea-pig liver*

“…Although more direct proof is required, our present results fit very well the scheme previously proposed (Francis & Ballard, 1980) for the inactivation of glucose 6-phosphate dehydrogenase and some other cytoplasmic enzymes. Accordingly, tyrosine aminotransferase appears to be inactivated in vitro by a disulphide-thiol exchange catalysed by a microsomal membranous factor.…”

A New Principle of Regulation of Enzymic Activity

“…Hence it becomes evident that activation of the latent collagenase might be linked to the phagocytizing activity of the cell and finally to its glucose metabolism. This system extends previous findings of intramolecular reductive processes in cellular regulation for the actimtion of glucose-6-phosphate dehydrogenase [23] and enzymic reactivation of pyruvate kinase [24] to bimolecular, oxidative ones. The protein-protein interaction of a proteolytic enzyme-inhibitor system is abolished during the disulfide-thiol interchange reaction upon which the active enzyme component with intact disulfide bridge is released.…”

“…In this case the oxidizing disulfide of the membrane protein converts two adjacent sulfhydryls of the active enzyme to an intramolecular disulfide in the inactive form. The activation of another enzyme, pyruvate kinase from rat liver, requiring free sulfhydryls in the active form is achieved by reduction of the glutathione disulfide [24]. In both cases the apparent molecular weights of the enzyme remain unchanged during the activation/inactivation process.…”

Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells

“…Post-translational modifications suggested to be determinative are ubiquitinylation (Ciechanover et al, 1984), oxidation (Levine et al, 1981) and deamidation (Robinson et al, 1970). Oxidative events that have been implicated in initiating extensive degradation include oxidation of essential histidine residues by mixed-function oxidation systems (Levine, 1983), attack by oxygen-derived free radicals (Wolff et al, 1986), and oxidation of cysteine residues by disulphides (Francis & Ballard, 1980;Offerman et al, 1984).…”