We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the twoelectron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k cat /K m ), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H 2 O 2 . An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.The enzyme pyranose oxidase (P2O) (pyranose:oxygen 2-oxidoreductase; EC 1.1.3.10), which catalyzes the oxidation of several aldopyranoses at position C-2 to yield the corresponding 2-ketoaldoses (aldos-2-uloses, osones), is widely distributed among wood-degrading basidiomycetes (14,32,44). It has been purified and characterized from several microorganisms, including Phanerochaete chrysosporium (2, 45), Phlebiopsis gigantea (39), Pleurotus ostreatus (40), Polyporus obtusus (28), Trametes (Coriolus) versicolor (35), and unidentified basidiomycete no. 52 (26). The currently available data reveal some general similarities among P2Os from these different fungi. Typically, P2O is a rather large, homotetrameric protein that contains covalently bound flavin adenine dinucleotide (FAD). The in vivo substrates of P2O probably are D-glucose, D-galactose, and D-xylose, which are abundant in lignocellulose and which are oxidized to 2-keto-D-glucose (D-arabino-hexos-2-ulose, 2-dehydro-D-glucose), 2-keto-D-galactose (D-lyxo-hexos-2-ulose, 2-dehydro-D-galactose), and 2-keto-D-xylose (D-threopentos-2-ulose, 2-dehydro-D-xylose), respectively. In addition, P2O also exhibits significant activity with a number of other carbohydrates, including L-sorbose, D-glucono-1,5-lactone, and D-allose (20). The substrate selectivity, however, varies to some extent among P2Os isolated from different fungi. During the oxi...

␥ -Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that ␥ -tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of ␥ -tubulin with ␣␤ -tubulin dimers. ␥ -Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large ␥ -tubulin complexes resistant to salt treatment were found to be associated with a highspeed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was Ͼ 1 MD. Large ␥ -tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate ␥ -tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of ␥ -tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of ␥ -tubulin suggests additional roles for this protein species in microtubule organization.

Electrical wiring of different types of pyranose oxidase (P2O) (fungal wild type, recombinant wild type with a hexahistidine tag, mutant form E542K with a hexa-histidine tag) from Trametes multicolor, and recombinant P2O from Coriolus sp. overexpressed in Escherichia coli as well as of pyranose dehydrogenase (PDH) from Agaricus meleagris and Agaricus xanthoderma with an osmium redox polymer (poly(1-vinylimidazole) 12 -[Os(4,4'-dimethyl-2,2'-dipyridyl) 2 -Cl 2 ] 2þ/þ ) on graphite electrodes was carried out. After optimization studies using glucose as substrate, the biosensors, which showed the best characteristics in terms of linear range, detection limit and sensitivity were selected, viz. wild type P2O from T. multicolor and PDH from A. meleagris. These two enzymes were used and investigated for their selectivity for a number of different sugars.