Abstract
Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.
Highlights
The Km values were derived from Lineweaver-Burk plots of the data
obtained using an enzyme assay system which contained the second substrate at the fixed concentrations indicated in the footnotes
Xanthine dehydrogenase activity was measured in crude extracts of N. crassa grown on
Summary
Neurospora cmssa (wild type STA4) was cultured as previously described [17]. Large scale growth for enzyme purification was carried out for 16 to 18 h following inoculation of 150 liters of liquid medium in a Biotec fermentor (capacity, 300 liters). The frozen mycelia were homogenized in 3 volumes of 0.1 M phosphate buffer, pH 8 per g of mycelia with a Ten Broeck tissue grinder This crude homogenate was centrifuged at 20,000 x g for 20 min and the supernatant, designated as the crude extract, was assayed for xanthine dehydrogenase activity by the fluorometric procedure (see below). The assay was performed in 0.1 M phosphate buffer, pH 8, containing 20 PM NAD+ and 10 PM xanthine or hypoxanthine. Protein samples containing 100 to 250 Fg of enzyme/ml were dialyzed overnight against deionized water. Gels were stained for protein with Coomassie blue R-250 and for xanthine dehydrogenase activity with a mixture of 10 mM hypoxanthine and 3 mM p-iodonitrotetrazolium violet or nitro blue tetrazolium in 0.1 M phosphate buffer, pH. Sepharose 4B, Sepharose 6B, and Se&adex were products of Pharmacia
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