Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

X-ray crystallographic characterisation of type-2-depleted ascorbate oxidase from zucchini.

The type-2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X-ray crystallography and EPR spectroscopy. The X-ray structure analysis by difference-Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR-active type-2 copper is depleted most; type-1 copper is not affected. This observation indicates preferential formation of a 1 Cu-depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu-depleted species may represent an anti-ferromagnetically coupled copper pair which is EPR-silent and could explain the disappearance of the type-2 EPR signal.     

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo).

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with 'high affinity' (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with 'low affinity' (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 g ml–1 blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 g ml–1 tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 g ml–1. After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 g ml–1 tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.     

Anaerobic reaction of ascorbate oxidase with ascorbate

Ascorbate oxidase is fully reduced by 4 mol of ascorbate in the absence of air, as monitored by optical and electron paramagnetic resonance spectra. At less than stoichiometric ascorbate concentration there is a slow equilibration between the 605-and 330-nm absorption bands: The 605-nm chromophore is first reduced, then its color reappears while the 330-nm absorption band decreases. Upon reoxidation with air the process takes place in the opposite direction. Intramolecular rather than intermolecular electron exchange appears to be responsible for this process. The reduced protein is about twice as fluorescent as the oxidized protein. The fluorescence quenching in the oxidized protein is related to the 330-nm absorption band rather than to the 605-nm band as previously reported for laccase.     

Reactivity of cytochrome oxidase with ascorbate

When cytochrome oxidase is solubilized in bile acid, ascorbate alone is capable of reducing the oxidase and induces oxygen uptake in the absence of cytochrome c. Cytochrome oxidase is organized into vesicular structures in the absence of detergent in phosphate buffer. In this, spectral changes are brought about by ascorbate, but there is negligible oxygen uptake in the absence of cytochrome c.     

Cytochrome oxidase and ascorbic acid oxidase activities in cereal plants

Cytochrome oxidase and ascorbic acid oxidase activities were investigated in rye, wheat, barley and oat plants. The variations in the activity of both enzymes was followed in the course of the initial 28 days of growth, as well as at the phase of milk ripeness, namely in the cytoplasmic and mitochondrial cell fractions of roots, leaves and spikes. Both enzymes were active in all measurements. Cytochrome oxidase mostly exhibited a higher activity than ascorbio acid oxidase. The activity of the former enzyme was substantially higher in the mitochondrial fraction of leaves, roots and spikes of the four experimental plants in comparison with the cytoplasmic fraction. On the contrary, the ascorbic acid oxidase activity varied in both cell fractions according to the plant species, organ and growth phase. The variations in the activity of both enzymes exhibited on the whole a course similar to that of the respiration rate. During the first 14 to 21 days of growth the enzyme activities increased up to the maximum. This was thon followed at first by a rapid, later on by a slow decrease. The course of variations in the enzyme activities was, with certain exceptions, alike in all the four plant species investigated.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Determination of inorganic phosphorus using immobilized pyruvate oxidase

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one-month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well-established method.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.