9313781 Massey We have succeeded in isolating milk xanthine dehydrogenase in a stable state, which can be converted reversibly into the oxidase form, where the preferred electron acceptor is 02, rather than NAD, as it is with the dehydrogenase form. Our work to date indicates that the major effects of this reversible interconversion, which is brought about by oxidation of spatially vicinal thiol residues in the dehydrogenase to oxidase conversion, involve conformational changes in the protein which affect mainly the flavin redox center. However, there are indications from rapid reaction studies that the molybdopterin redox center may also be affected by the interconversion. The major thrust of this proposal is to study in detail the physicochemical properties of the two forms of the enzyme, with the aim of defining the parameters that give the two forms such different catalytic properties. This study will involve extensive use of rapid reaction spectrophotometry, EPR and EXAFS measurements, flavin replacement studies, catalytic properties of the enzymes with different classes of substrates, and examination of the modes of binding of potent inhibitors of the enzymes. %%% The enzyme xanthine oxidase is commonly believed to be implicated in the phenomenon known as oxidative stress, in which free radical species of oxygen react with many essential components of living tissues, including proteins, lipids and DNA. Such oxidative damage has been implicated in cancer, ischemia and the aging process. Xanthine oxidase exists in two forms, the oxidase, which oxidizes xanthine and a wide variety of purines and pyrimidines at the expense of reducing oxygen to the superoxide radical (02-), and the dehydrogenase, which oxidizes the same compounds, but uses NAD, a coenzyme derivative of vitamin B3, instead of oxygen. We have isolated the enzyme from cow's milk, and shown that the two forms can be interconverted in a reversible manner, depending on the oxidation-reduction state of the protein thiol residues. The two enzyme forms have many different properties, which are clearly due to different protein conformations. While the oxidase form has been studied extensively in the past, comparatively little is known of the dehydrogenase. This study is to explore in detail the differences in properties of the two forms, in order to provide a firm scientific basis for concepts regarding their physiological functions. ***
