Hepatic xanthine dehydrogenase. I. Purification and protperties of the human and pig hepatic xanthine dehydrogenase

Abstract

The xanthinedehydrogenase activity of human and porcine liver homogenates has been found to be located in the supernatants, when the particulated cell fractions have been previously separated by diferential centrifugation, whereas uricase appears to be associated with the mitochondrial fraction.

When the soluble proteins of the he­ patic homogenates are submitted to a fractional ammonium sulphate precipitation, xanthinedehydrogenase accumulates in the sediment obtained with a salt concentration of O.3-O.6 saturation. The human enzyme can be further purified by means of a DEAE-Cellulose chromatographic separation, when its specific activity increa­ses up to 65 times. Pig liver xanthinedehydrogenase has been purified first by a fractional precipitation with ammonium sulphate, followed by a controlled tryptic digestion and a chromatographic adsorption in a DEAE-Sefadex column: the enzymatic preparation thus obtained is about 152 times concentrated.

Both purified preparations show spectral absorption curves similar to chicken and calf liver xanthinedehydrogenases, and their extinction relationships at 280/ 450 m/x and 410/450 m/x, indicate a reasonable degree of purification of these enzymatic proteins.

The Michaelis-Menten constants of both enzymes are: pig liver xanthinede-hydrogenase, Km (O2) = 2.28 X 10-5 M; Km (M. B.) = 2.7 X 10-i M; human liver xanthinedehydrogenase, Km (M. B.) = 3.1 X 10-5 M. Colchicine is a competi­tivo inhibitor of both dehydrogenases: pig Kx (O.) = 1.22 X 10"231M; Kx (M. B.) = 1.14 X10-3 M; human Kx (M. B.) = 2.5 X 10-3 M.