Electrophoretic and Chemical Studies on the X-Ray Damage of Malate Synthase

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Abstract
The sulfhydryl enzyme malate synthase (baker''s yeast) was shown to undergo an X-ray induced aggregation and inactivation in solution. Further evidence for the occurrence of aggregation and inactivation and also of fragmentation and partial unfolding of the enzyme upon X-irradiation was obtained by chemical and electrophoretic studies. Irradiation was carried out in a specially designed microcell, experiments were performed on the microlevel. Under conditions of the experiments the formation of H2O2 upon X-irradiation could be proven; therefore the influence of H2O2 on the enzyme was investigated too. Though the quantitative results of the damaged enzyme particles are influenced by many distrubing factors, the findings allow clear statements on the nature of the effects under investigation. Both X-irradiation and treatment with H2O2 caused a decrease of total and an increase of available sulfhydryl groups of the enzyme and led to a loss of enzymic activity. The presence of dithiothreitol turned out to be able to protect the enzyme against X-ray or H2O2 induced inactivation. Addition of dithiothreitol after X-irradiation or H2O2 treatment allowed a considerable repair of enzymic activity. Polyacrylamide gel disc electrophoreses of X-irradiated enzyme solutions, performed in the presence of sodium dodecyl sulfate, showed the occurrence of covalently cross-linked subunits (preferably dimers and trimers) and of various definite fragments. Electrophoreses in the absence of the denaturant indicated the occurrence of enzyme aggregation. The effects were more pronounced with increasing X-ray doses. The electrophoreses also clearly reflected a radioprotection by dithiothreitol against cross-linking, but not against fragmentation. Addition of excess of 2-mercaptoethanol or of dithiothreitol to the X-irradiated enzyme clearly demonstrated that part of the covalent cross-links were disulfide bridges; the aggregated themselves were held together primarily by non-covalent bonds. Blocking of exposed enzyme sulfhydryls by means of Ellman''s reagent prevented both covalent cross-linking and enzyme aggregation. Similar electrophoretic patterns as found for the X-irradiated enzyme were obtained for the unirradiated enzyme after treatment with H2O2. The similarity of the electropherograms, as well as the reversible diminution of enzymic activity and the loss of sulfhydryls in the presence of H2O2, suggest an involvement of H2O2 in the radiation damage of the enzyme. It seems plausible that oxidation reactions are responsible for the effects caused by X-irradiation or H2O2 treatment.