Mitochondrial Malate Dehydrogenase of Bovine Cerebrum

Characterization and Mechanisms of Inhibition by Silver Ions

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Attempts were made to characterize mitochondrial malate dehydrogenase [L-malate: NAD⁺ oxidoreductase, EC 1. 1. 1. 37] (M-MDH) purified from bovine cerebrum and to elucidate the mechanisms responsible for inhibition of the enzymic activity by Ag⁺. The molecular weights of the native enzyme and its subunits were 54, 000-55, 000 and 30, 000-32, 000, respectively. In general, the physicochemical and catalytic properties of bovine cerebral M-MDH was not very different from those of other corresponding mammalian enzymes. Incubation of the enzyme with Ag⁺ caused the loss of equivalent amounts of sulfhydryls with a parallel decrease of the enzymic activity. When the enzyme was exposed to 2-, 3.5-, and 5-fold molar excesses of Ag⁺, the enzymic activity showed an initial rapid fall and a subsequent slow restoration to a partially inactivated level (60-70, 45-50, and 15-20% of an untreated control, respectively), while the α-helical content of the enzyme fell exponentially with time. A 7-fold molar excess of Ag⁺ reduced both the enzymic activity and the α-helical content to a much greater degree and no restoration of the enzymic activity was observed. The K_m values of Ag⁺-inactivated enzyme for NADH and oxaloacetate were the same as those of the native enzyme. The data suggest that Ag⁺ could inhibit the enzymic activity both by reducing the structural regularity of the enzyme molecule and by attacking sulfhydryl groups necessary for the catalytic activity of bovine cerebral M-MDH.