Abstract
Disulfides (glutathione disulfide, cystine, cystamine) caused a first-order inactivation of rabbit-muscle fructose-1,6-bisphosphate aldolase at pH values of 7.4 and above. Inactivation by glutathione disulfide was partially reversed by reducing agents, but the enzyme became irreversibly inactivated with time. The disulfide-inactivated aldolase had a lower transition temperature and enthalpy of denaturation than the native enzyme. In addition, the disulfide-inactivated enzyme was extensively degraded by proteinases, whereas the native enzyme was resistant. Mixed disulfides were formed; a maximum ratio of 4-5 mol of glutathione/mol of the aldolase tetramer was found. The number of titratable--SH groups on aldolase decreased by 16 (out of 32 total on the control enzyme) after inactivation by glutathione disulfide, indicating that other oxidation reactions in addition to those resulting in mixed disulfides occurred. The substrate, fructose 1,6-bisphosphate, prevented inactivation of aldolase by glutathione disulfide, the formation of glutathione-enzyme mixed disulfides, thermodynamic destabilization of the enzyme, and a decrease of--SH groups on the enzyme. These data indicate that covalent modification of aldolase by biological disulfides is important in modulating enzyme stability and vulnerability to proteinases as well as enzyme activity and that the substrate protects against modification by disulfides.
Highlights
Disulfides initiate the degradation of specific liver proteins
The native enzyme is readily inactivated in vitroby severalproteases, only the C-terModulation of enzyme activity by covalent modification is minal region of the native form is well documented, and the importance of this phenomenon in readily vulnerable to proteolysis; this limited proteolysis biological regulation is established
In order to determine whether the formation of mixed disulfides was essential for the observed change in aldolase proteolytic susceptibility, aldolase was treated with sodium periodate
Summary
Matography, and theprotein concentration and activity were determined aspreviously described [18]. Droxyacetone 3-phosphate (4mM), a product of the aldolasecatalyzed reaction, had no effect on the rate of inactivation of the enzyme by disulfides (data not shown). Aldolase inactivation by glutathione disulfide was partially reversed by dithioerythritol but became partially irreversiblewith time. The filtrate inactivation of aldolase with 12 mM glutathione disulfide at was concentrated by lyophilization and subjected to Sephadex G-10 pH 8.0 ranged from 15 to 30 min on different days, an column (1.5 X 25 cm) chromatography. At concenprepared in theabove manner inactivated aldolase at a predictable rate, from parallel experiments with authentic glutathione disulfide, and could be reduced by glutathione reductase and NADPH in 50 mM Tris-HC1,pH 7.5, containing 1mM EDTA as measured by DTNB.
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