Suppression of rabbit myocardial xanthine dehydrogenase activity by an endogenous compound

Xanthine dehydrogenase (XDH) is an important precursor to the oxygen radical producing enzyme xanthine oxidase (XO). We found that the apparent activity of rabbit myocardial XDH increased from 2 ± 1 to 50 ± 3 μU/g (P<0.05) following extraction of tissue homogenate with butanol. Further studies suggested that the basis for this observation was a high molecular weight compound which consumes the XDH cofactor, NAD⁺. Addition of myocardial homogenate to exogenous NAD⁺ resulted in depletion of NAD⁺ and concomitant formation of an additional compound (peak A). Both NAD⁺ consumption and peak A formation were abrogated by prior extraction of homogenate with butanol. Separation of myocardial homogenate by Sephadex chromatography revealed a high molecular weight compound which suppressed activity of purified milk XDH but not xanthine oxidase (XO). This activity co-eluted with the ability of myocardial homogenate to consume added NAD⁺ and form peak A. The NAD⁺-consuming activity was heat and acid-labile. In addition, nicotinamide was both a product and an inhibitor of the NADase activity, consistent with the existence of a previously described myocardial glycohydrolase. Extraction of tissue with butanol may be necessary to detect low levels of XDH activity in vitro.