ORGAN DISTRIBUTION AND MOLECULAR-FORMS OF HUMAN XANTHINE DEHYDROGENASE XANTHINE-OXIDASE PROTEIN

Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmi c source of superoxide radicals and hydrogen peroxide, and it is consi dered important in the pathogenesis of ischemia-reperfusion damage. Be cause little is known about the enzyme in human tissues, the aims of t his study were to purify human XDH/XO and to produce Ab for detection of the protein in Western blots and for quantification by ELISA. We pu rified human milk XDH/XO, produced Ab for Western blotting and ELISA o f the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified pro tein under nondenaturing conditions was approximately 300 kd. On SDS-P AGE, it was fragmented into four main bands of 143, 125, 87, and 59 kd . Ab recognized bands of similar size in Western blots of the purified preparation and human milk. In fresh liver homogenates treated with a nti-proteases, the three largest bands were observed; in the intestine , only the two largest were observed. Serum, brain, heart, and skeleta l muscle were negative, whereas some lung and kidney samples showed on e faint band of 143 kd. Trypsin treatment of the enzyme converted the large molecular-weight bands into smaller bands, as did incubation of a liver homogenate without anti-proteases. XDH/XO protein concentratio ns (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in i ntestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mu mol/min/mg XDH/XO protein) was constant in live r and intestine during development. We conclude that 1) human XDH/XO h as molecular size and subunit structure similar to other mammalian enz ymes; 2) the polypeptide chain is unstable, also in the intact cell, d espite retained activity; and 3) the amount of inactive XDH/XO in huma n liver and intestine is apparently small.