The inhibition of xanthine and succinic oxidases by carbonyl reagents.

Previous studies have demonstrated the existence of three different types of enzymes that oxidize santhine. The enzyme found in bird tissues is a dehydrogenase without significant ability to react directly with oxygen (1) ; it can be assayed manometrically by the use of methylene blue as an electron carrier between the reduced enzyme and oxygen. The enzyme found in mammalian tissues reacts directly with air and is therefore an oxidase like milk xanthine oxidase (2). These two oxidases can be differentiated by the fact that only the tissue enzyme can be inhibited by tetraethylthiuram disulfide (Antabuse) (3) or chalcones (4), while the milk enzyme is unaffected. This inhibition of the tissue enzyme by Antabuse or chalcones is limited to its oxidase activity, and no effect is observed on its dehydrogenase activity with methylene blue as the electron acceptor. The three enzymes are therefore fundamentally alike in catalytic activities, but presumably differ in that portion of the molecule responsible for the reaction with air. All three enzymes have been obtained in highly purified form (1, 5, 6), and each contains Fe, flavin adenine dinucleotide, and MO in the ratios 8 :2: 1, 4 : 1: 1, and 8 : 1: 1 for the milk, mammalian liver, and chicken liver enzymes, respectively. A comparison of these relative compositions cannot yet account for the differences in reactivity with oxygen (7). It is possible that the two xanthine oxidases contain an additional unidentified “oxidase” group, such as a quinone, which is absent from the dehydrogenase. The present study demonstrates that the oxidase activity of the enzymes from milk and from rat liver can be inhibited selectively by the carbonyl reagents, phenylhydrazine, and semicarbazide; this inhibition can be reversed completely by 2-methyl-1,4naphthoquinone. However, glutathione was also effective in overcoming the semicarbazide inhibition, and chelating agents (e.g. 8-hydroxyquinoline) were effective under certain conditions. The latter effects implicate the enzymatic iron as the point of attachment by the carbonyl reagents. Numerous studies in recent years have implicated a quinone in the mitochondrial electron transport chain from DPNH or succinate to oxygen. Some of these studies point to a quinone related to vitamin K (8-l 1) and others to a quinone derived from ol-tocopherol (12-14). A quinone related to vitamin K was recently isolated from Mycobacterium phlei, and was shown to restore oxidative phosphorylation to an irradiated bacterial preparation (15). The only quinone isolated (16, 17, 14) from