A spectrophotometric method for the determination of three forms of xanthine oxidoreductase, namely dehydrogenase (D), dehydrogenase-oxidase (D/0) and oxidase (0)

Data processing using nonparametric statistics was performed using a 'direct linear plot' algorithm of ECB. ECB (Eisenthal and Cornish-Bowden) is a computer program designed to calculate the Km and V parameters in enzyme kinetics. It is also suitable for estimation of EC50 (ligand or drug concentration for 50% effect) in pharmacological studies.

The effects of 2-iodosobenzoic acid, 4-chloromercuribenzoate, 5,5'-dithiobis-(2-nitrobenzoic acid) and tetraethylthioperoxydicarbonic diamide (disulphiram) on the NAD+-dependent activity of xanthine oxidoreductase from rat liver were investigated. Only disulphiram converted the NAD+-dependent activity into the O2-dependent activity quantitatively, without changing the xanthine hydroxylation rate. The modification process was a first-order reaction with respect to time (min) and disulphiram concentration (microM). The kinetic data showed that modification of single thiol group is sufficient for loss of the enzymic activity towards NAD+ as electron acceptor. The complete protection afforded by NAD+ against the action of disulphiram suggests that the essential thiol group may be involved in binding of NAD+ to the xanthine oxidoreductase molecule.

Abstract. The Astro Izery project is carried by several institutions from Poland and Czech Republic. Its aim is to educate and inform tourists, who visit the Izery Mountains, about astronomy and light pollution. The project consists of two activities: permanent (sundials, planetary path etc.) and periodic (meetings, workshops). After five years the project is in good health and will gain more elements in next years.

The enzyme xanthine: acceptor oxidoreductase found in rat heart equilibrates between three forms differing in electron acceptor specificity. Form D transfers electrons exclusively to NAD+ and accounts for 85% of total oxidoreductase activity. Form O transfers electrons to molecular oxygen and accounts for 8%. The D/O form prefers NAD+, but without NAD+ transfers electrons to oxygen. Interconversion from D to O and O to D forms is catalyzed by sulfhydryl group-modifying reagents: Cd2+, Cu2+, disulfiram, and heating with dithiothreitol. This suggests that sulfhydryl groups participate in the first stage of enzyme conversion. The NADH/NAD+ concentration ratio may regulate the dehydrogenase activity of xanthine:acceptor oxidoreductase (NAD+-dependent activity of D and D/O forms). Accumulating NADH inhibits hypoxanthine hydroxylation. The amount of form O increases during cardiac ischemia, facilitating superoxide radical-ion generation. Also, NADH/NAD+ does not regulate form O, promoting adenylate nucleotide pool depletion, especially in the heart which has low de novo purine nucleotide synthesis.