Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol.
Although the binding of xanthine oxidase (XO) to glycosaminoglycans (GAGs) results in significant alterations in its catalytic properties, the consequence of XO/GAG immobilization on interactions with clinically relevant inhibitors is unknown. Thus, the inhibition kinetics of oxypurinol for XO was determined using saturating concentrations of xanthine. When XO was bound to a prototypical GAG, heparin-Sepharose 6B (HS6B-XO), the rate of inactivation for uric acid formation from xanthine was less than that for XO in solution (k(inact) = 0.24 versus 0.39 min(-1)). Additionally, the overall inhibition constant (K(i)) of oxypurinol for HS6B-XO was 2-5-fold greater than for free XO (451 versus 85 nm). Univalent electron flux (O(2)(.) formation) was diminished by the binding of XO to heparin from 28.5% for free XO to 18.7% for GAG-immobilized XO. Similar to the results obtained with HS6B-XO, the binding of XO to bovine aortic endothelial cells rendered the enzyme resistant to inhibition by oxypurinol, achieving approximately 50% inhibition. These results reveal that GAG immobilization of XO in both HS6B and cell models substantially limits oxypurinol inhibition of XO, an event that has important relevance for the use of pyrazolo inhibitors of XO in clinical situations where XO and its products may play a pathogenic role.
Kelley
Eric E
EE
Department of Anesthesiology, and the Center for Free Radical Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. ekelley@uab.edu
Trostchansky
Andrés
A
Rubbo
Homero
H
Freeman
Bruce A
BA
Radi
Rafael
R
Tarpey
Margaret M
MM
D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000378:metabolism*; D007700:Kinetics; D010117:Oxypurinol / Q000494:pharmacology*; D011485:Protein Binding; D013481:Superoxides / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D013481:Superoxides; D014527:Uric Acid; D014969:Xanthine Oxidase; D010117:Oxypurinol