Reexamining Michaelis-Menten enzyme kinetics for xanthine oxidase.

Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions are violated. Here, we analyzed data of xanthine oxidase in vitro from Escribano et al. (Biochem J 254: 829, 1988) on two conversions in series, hypoxanthine to xanthine to uric acid. Analyses were done using four models: standard irreversible M-M reactions (model 1), Escribano et al.'s M-M forward reaction expressions with product inhibition (model 2), fully reversible M-M equations (model 3), and standard differential equations allowing forward and backward reactions with mass balance accounting for binding (model 4). The results showed that the need for invoking product inhibition vanishes with more complete analyses. The reactions were not quite irreversible, so the backward reaction had a small effect. Even though the enzyme concentration was only 1-2% of the initial substrate concentrations, accounting for the fraction of solutes bound to the enzyme did influence the parameter estimates, but in this case, the M-M model overestimated Michaelis constant values by only about one-third. This article also presents the research and models in a reproducible and publicly available form.

Effects of Greek legume plant extracts on xanthine oxidase, catalase and superoxide dismutase activities

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Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered (AU)

Tumor-targeting chemotherapy by a xanthine oxidase-polymer conjugate that generates oxygen-free radicals in tumor tissue.

Xanthine oxidase (XO) mediates anticancer activity because of its ability to generate cytotoxic reactive oxygen species (ROS), including superoxide anion radical and hydrogen peroxide. However, the high binding affinity of XO to blood vessels would cause systemic vascular damage and hence limits the use of native XO in clinical settings. We demonstrate here that chemical conjugation of XO with poly(ethylene glycol) (PEG; the conjugates hereafter referred to as PEG-XO) significantly enhanced the tumor-targeting efficacy and the antitumor activity of XO. By using a succinimide-activated PEG derivative, PEG was conjugated to epsilon-amino groups of lysine residues of XO, which play a crucial role in binding of XO to blood vessels. PEG-XO administered i.v. showed a 2.8-fold higher accumulation in solid tumor compared with that of native XO 24 h after injection, whereas a slight or negligible increase in accumulation of PEG-XO was observed in normal organs. The highest PEG-XO enzyme activity was detected in tumor compared with normal organs or tissues except blood; enzyme activity in tumor was 5.0, 3.9, and 9.4 times higher than that in liver, kidney, and spleen, respectively. Intratumor activity remained high for >48 h. Administration of hypoxanthine, a substrate of XO, at 33 mg/kg body weight i.p. 12 h after the administration of PEG-XO (0.6 unit/mouse, i.v.) resulted in significant suppression of tumor growth (P < 0.001), with no tumor growth even after 52 days. However, either PEG-XO or hypoxanthine alone, or native XO with hypoxanthine, showed no effect on the inhibition of tumor growth under present experimental conditions. These findings suggest that PEG-XO, which accumulates preferentially in tumor tissue, warrants further investigation as a novel anticancer agent.

Tissue distribution of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in male and female guinea pigs.

The activity of the molybdenum hydroxylase, aldehyde oxidase, was determined in crude homogenates and (NH4)2SO4 fractions prepared from guinea pig liver, lung, kidney, intestine, spleen and heart. Xanthine oxidase was also measured in (NH4)2SO4 fractions. In each case, xanthine oxidase levels were lower than those of aldehyde oxidase; activity of the latter enzyme was highest in the liver, whereas xanthine oxidase was predominant in the small intestine. There was no significant difference in the activity of either molybdenum hydroxylase between tissues taken from male and female guinea pigs.