A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (MJ of 322800 It 18300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDSPAGE resulted in one single polypeptide band of Mr76000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-galactose, methyl P-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-~-glucose, 5-keto-~-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy I3C-NMR spectroscopy and 'H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0-6.5 in 0.1 M potassium phosphate, and its activation energy ( A H") for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the K, values determined for D-glucose, L-sorbose, D-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.

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1. The intermediate structures formed during dialysis of mixtures of cholate, phospholipid and cytochrome c oxidase were analysed by gel chromatography and electron microscopy. Measurements of trapped phosphate and the degree of respiratory control were used to assess the integrity of the vesicular structures formed. Protein orientation in the bilayer was monitored by the accessibility of cytochrome c to cytochrome c oxidase. 2. The results indicate that proteoliposome formation by the detergent-dialysis procedure takes place in three distinct stages. In the first stage, cholate/phospholipid and cholate/phospholipid/protein micelles coexist in solution and grow in size as the detergent is slowly removed. At a detergent/phospholipid molar ratio of about 0.2, micelle fusion results in the formation of large bilayer aggregates permeable to both phosphate and cytochrome c. It is at this stage that cytochrome c oxidase is incorporated into the bilayer. In the final stage of dialysis the bilayer sheets fragment into small unilamellar vesicles. 3. The orientation of membrane protein in the final vesicles appears to be determined by the effect of protein conformation on the initial curvature of the bilayer sheets during the fragmentation process.