Synthesis of xanthine oxidase by rat liver slices in vitro.

The influence of dietary protein on the activity of rat liver xanthine oxidase has been studied by several workers (l-5) and would point to a relationship between the availability of amino acids and the activity of liver xanthine oxidase (2). It has been demonstrated that the loss of liver xanthine oxidase activity in the rat greatly exceeds the loss of liver protein during acute inanition (l), revealing thereby the labile nature of this enzyme. This apparent functional lability of rat liver xanthine oxidase is further indicated in the results reported by Litwadk et al. (5) and would suggest that the enzyme is capable of easy synthesis under favorable conditions. In accordance with the view of the participation of enzymes in the actively metabolizing pool of proteins, Anfinsen (6) isolated radioactive crystalline ribonuclease from bovine pancreas slices that had been incubated with Cl40 2. Very recently, Hokin (7) has reported on the synthesis in vitro of amylase in amylase-depleted pigeon pancreas slices by incubating the slices in media containing the essential amino acids. As far as is known, these are the only instances in which synthesis of an enzyme in vitro has been established. It was thought that a demonstrable increase in xanthine oxidase act.ivity might result upon incubation of liver slices from protein-depleted rats in media containing essential nutrients; the stimulus to recovery of liver substance should be expected to be more potent the more severe its reduction (8,9). A prerequisite to this study was an accurate method for following xanthine oxidase activity. The manometric method of Axelrod and Elvehjem (10) presented difficulty owing to the high endogenous respiration as well as to inhibition of endogenous respiration by added xanthine. However, preliminary incubation for 40 minutes or dialysis at 0” for 18 hours could, as suggested by the authors, remove endogenous purine substrates without impairment of xanthine oxida-se activity. Precipitation of the enzyme (11) to free it from endogenous substrates was not always satisfactory. The procedure of Dixon and Thurlow (12), in which anaerobic reduction of nitrate by xanthine oxidase is employed, was found to be non-specific. The coupling of the oxidation of xanthine to uric acid with the reduction of oxidized cytochrome c has been made the basis of a method for measuring xanthine oxidase activity through spectrophot.ometric study