Organ distribution and molecular forms of human xanthine dehydrogenase/xanthine oxidase protein.
Xanthine dehydrogenase/xanthine oxidase (XDH/XO) is a major cytoplasmic source of superoxide radicals and hydrogen peroxide, and it is considered important in the pathogenesis of ischemia-reperfusion damage. Because little is known about the enzyme in human tissues, the aims of this 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 purified human milk XDH/XO, produced Ab for Western blotting and ELISA of the protein, and evaluated the molecular forms and activity-protein relationships in human tissues. The molecular size of the purified protein under nondenaturing conditions was approximately 300 kd. On SDS-PAGE, 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 anti-proteases, the three largest bands were observed; in the intestine, only the two largest were observed. Serum, brain, heart, and skeletal muscle were negative, whereas some lung and kidney samples showed one 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 concentrations (ng/mg total protein) were 146 +/- 70 in liver and 556 +/- 320 in intestine and less than 5 ng/ml in serum. The relationship of activity to protein (2.7-3.0 mumol/min/mg XDH/XO protein) was constant in liver and intestine during development. We conclude that 1) human XDH/XO has molecular size and subunit structure similar to other mammalian enzymes; 2) the polypeptide chain is unstable, also in the intact cell, despite retained activity; and 3) the amount of inactive XDH/XO in human liver and intestine is apparently small.
Sarnesto
A
A
Children's Hospital, University of Helsinki, Finland.
Linder
N
N
Raivio
K O
KO
D000328:Adult; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D010450:Endopeptidases; D005312:Fetal Blood / Q000201:enzymology; D005333:Fetus; D006801:Humans; D006868:Hydrolysis; D007106:Immune Sera / Q000737:chemistry; D007231:Infant, Newborn; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D013329:Structure-Activity Relationship; D014968:Xanthine Dehydrogenase / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D014969:Xanthine Oxidase / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification
D007106:Immune Sera; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D010450:Endopeptidases