Liver Xanthine Oxidase INVOLVEMENT OF THIOL GROUPS IN THE CONVERSION OF THE ENZYME ACTIVITY FROM DEHYDROGENASE (TYPE D) INTO OXIDASE (TYPE 0) AND PURIFICATION OF THE ENZYME By E. DELLA CORTE and F. STIRPE

1. The 'xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD+-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type 0) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type 0 brought about by storage of rat liver supernatant at -20°C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type 0 is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from typeD into type 0 during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol.