Stimulation of Ascorbate Oxidase secretion from cultured pumpkin cells by divalent cations

Abstract Ascorbate Oxidase is actively secreted during the growth of cultured pumpkin (Cucurbita spp.) cells. Calcium ions markedly stimulated the accumulation of Ascorbate Oxidase in the culture medium. Ascorbate Oxidase activity in the culture medium at 20 days after transfer to a fresh medium containing 40 mM calcium chloride (CaCl2) was about 50 times the level attained in the absence of CaCl2. Furthermore, the specific activity of the enzyme in the culture medium was increased ca 20-fold by adding 40 mM CaCl2. On the other hand, Ca2+ had little effect on the accumulation of perOxidase in the medium. Magnesium ions, but not K+, was shown to be effective in the stimulation of Ascorbate Oxidase secretion, suggesting that Mg2+ can substitute for Ca2+ in stimulating the secretion of Ascorbate Oxidase.

Secretion of Ascorbate Oxidase by suspension-cultured Pumpkin cells

Abstract Ascorbate Oxidase is released into the medium in pumpkin ( Cucurbita sp.) cell suspension cultures. Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase, cytosolic enzymes, and catalase, microbody enzyme, were not detected in the medium, whereas perOxidase, which is known to be a secretory protein, was detectable. Immunological blotting showed that the M r of Ascorbate Oxidase secreted into the medium was identical to that of the enzyme purified from pumpkin fruit tissue. Purified Ascorbate Oxidase on acrylamide gel was stained by periodic acid-Schiff method. Thus, Ascorbate Oxidase is probably a secretory glycoprotein.

Stimulation of Ascorbate Oxidase secretion from cultured pumpkin cells by eosine yellowish and potassium salicylate.

Abstract Ascorbate Oxidase is actively secreted during the growth of cultured pumpkin ( Cucurbita spp.) cells. Eosine Yellowish, which is known to be an inducer of pathogenesis-related proteins, markedly stimulated the accumulation of Ascorbate Oxidase in the culture medium. Potassium salicylate, another inducer of pathogenesis-related proteins, also stimulated the secretion of Ascorbate Oxidase. Thus, there is a possibility that Ascorbate Oxidase is a kind of pathogenesis-related proteins.

Preliminary crystallographic data for the copper enzyme Ascorbate Oxidase

Ascorbate Oxidase (EC 1.10.3.3) is a copper enzyme belonging to the group of so-called ‘blue Oxidases’ together with laccases and ceruloplasmin. The enzyme, widely distributed in several plant species, catalyzes the oxidation of L-Ascorbate, transferring the reducing equivalents to molecular dioxygen. The biological function of the enzyme is still in question. Ascorbate Oxidase activity is highest in those parts of plants which grow faster; on the other hand some authors suggested a possible role of the enzyme in plant respiration [1].

The native enzyme is a non-covalent dimer, whose subunits (respectively 75,000 and 72,000 Mr) contain 8 Cu2+ ions; these can be classified, according to their coordination environments, as of type-1, type-2 and type-3 [2, 3]. Ascorbate Oxidase is known to undergo fully reversible association-dissociation phenomena. Its ultracentrifuge pattern changes as a function of pH and buffer media, while the spectroscopic properties and the activity towards Ascorbate remain unchanged.

Although the information available at present is not sufficient to fully elucidate the sequence of redox events which take place within the protein, there exist some evidence that the three classes of copper ions fulfil different functions. Type-1 copper is the primary site of electron acceptance; type-2 and type-3 coppers are implicated respectively in Ascorbate and O2 binding [4]. Ascorbate Oxidase is thus an ideal model enzyme for the study of biochemistry and biophysics of vegetal copper proteins. In consideration of its physico-chemical properties, the elucidation of Ascorbate Oxidase three-dimensional structure may also contribute to the comprehension of the association-dissociation phenomena and of their biological significance.

The protein employed for the crystallization experiments was purified from green zucchini squash according to the method of Avigliano et al. [5], showed absorption ratios A280/A610 = 25 ± 1, A330/ A610 = 0.8 ± 0.05 and had a turnover number of 5 × 105 mol/min. Several micro-buttons filled with a 15 mg/ml solution of the enzyme were used in parallel dialyses experiments against different buffers and precipitating agents, in the pH range 5–9. Under the following conditions the same characteristic blue crystals of the enzyme could be grown:
1.
1.8 M ammonium sulfate, at pH values 6.7 through 8.4

2.
1.0 M sodium citrate, at pH 7

3.
1.9 M potassium phosphate, at pH 7

The crystals obtained were subsequently used for a preliminary crystallographic. From the analysis of the diffraction pattern symmetry and of the systematic absences it was possible to conclude that Ascorbate Oxidase crystallizes in the orthorhombic space group P212121 with unit cell edges a = 125.4, b = 189.8, c = 112.2 A. The asymmetric unit can thus accommodate a dimer of the enzyme (Mr 294,000) and the (volume) solvent content of the crystals is 45%. The crystals diffract to 3.0 A resolution; this crystalline modification is isomorphous with that reported by Ladenstein et al. [6] for the same enzyme, but grown under different physico-chemical conditions.

Removal of type 2 Cu from Ascorbate Oxidase and laccase by reaction with n,n-diethyldithiocarbamate

Abstract The type 2 Cu of Ascorbate Oxidase from zucchini peelings can be rapidly removed by reaction with a tenfold excess N,N -diethyldithiocarbamate (DDC) in air, while other chelating agents, such as EDTA, require anaerobic reducing conditions. The type 2 Cu of laccase from Rhus vernicifera is never removed under aerobic conditions. In anaerobiosis and in the presence of a reducing agent, EDTA is also unable to remove the copper unless a smaller lipophilic molecule (DDC or dimethylglyoxime) is present, acting as a mediator. Type 1 Cu is not involved in the reaction of Ascorbate Oxidase with DDC, but reduction of type 3 Cu is probably required for type 2 Cu depletion, suggesting interdependence of type 2 and type 3 copper. Type 2 Cu is less exposed in laccase, possibly because of the large carbohydrate content of this protein.

Exhaustive removal of N-glycans from Ascorbate Oxidase: effect on the enzymatic activity and immunoreactivity

Purified Ascorbate Oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active Ascorbate Oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native Ascorbate Oxidase antibodies scarcely recognized deglycosylated protein.

Spectral study of Ascorbate Oxidase

Abstract Ascorbate Oxidase (L-Ascorbate:O 2 oxidoreductase, EC 1.10.3.3) is the most complex member of the group of enzymes known as the blue copper Oxidases [1]. The protein contains the three types of biological copper, according to the Malmstrom classification [2], in the stoichiometry of three type 1, one type 2 and four type 3 copper atoms per molecule. We have undertaken an investigation of the spectral properties of Ascorbate Oxidase isolated from the green zucchini squash and wish to present here our preliminary results. The protein was purified according to the most recently published procedure [3]. The parameters M r = 140 000 and ϵ 610 = 9700 M −1 cm −1 were assumed, while the ratio A 330 /A 610 for our preparation was approximately 0.90. The X-band EPR spectrum of a frozen solution of Ascorbate Oxidase in 0.1 M phosphate buffer, pH 6.8, recorded at −140 °C, was fitted according to the following computer simulation procedure. We minimized the sum of errors Z = ∑ i [G exp (H i ) − G th (H i )] 2 where G(H) is the line-shape function sampled at 220 discrete points of the field. The theoretical line-shape was considered as a sum of the type: G th = α 1 G th 1 (g z , g x , g y , A z , A x , A y ) +α 2 G th 2 (g ∥ , g ⊥ , A ∥ , A ⊥ ) where α 1 and α 2 are the molar fractions and G th 1 andG th 2 the theoretical spectra of the type 1 and type 2 copper species. The lowest accuracy (±2%) in the parameter determination is that relative to α 1 and α 2 , due to a rather smooth variation of the Z value with respect to these parameters. The best fit of the EPR spectrum was obtained with the following parameters: p]The molar fractions of type-1 and type-2 copper were estimated as 0.75 ± 0.02 and 0.25 ± 0.02, respectively. Double integration of the EPR signal revealed that 49.5% of the total copper was EPR-detectable. Our data are therefore in close agreement with those reported earlier for similar preparations of Ascorbate Oxidase [3, 4]. The visible CD spectrum of Ascorbate Oxidase displays extrema at 735 (Δϵ = −15.85 M −1 cm −1 , 610 (+6.97), 475 (−4.85) and 330 nm (−2.08). Additional very weak positive activity may occur near 420, though this is actually often indistinguishable from zero. While the magnitude of the Cotton effects within these visible CD bands is similar to that reported by Gray [5], the location of the extrema occurs at slightly different wavelengths. In the near-UV region the CD spectrum of Ascorbate Oxidase is mainly contributed by the aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) and by the disulfide bonds of cystine residues, while minor contributions may also be expected to arise from copper(II)-ligand charge transfer transitions. The near-UV maxima are located near 296 (+24.6), 291 (+28.4), 283 (+39.7) and 265 nm (+60.5, broad), while additional negative CD activity near 240 nm appears as a shoulder on the protein CD bands at higher energy. We note that the near-UV absorption spectrum of Ascorbate Oxidase is dominated by an intense band centered at 280 nm with shoulders near 290 and 260 nm. The far-UV CD spectrum between 200 and 240 nm contains a strong negative protein band at 218 nm (θ = −16700 deg cm 2 dmol −1 where θ is the mean residue ellipticity calculated on the basis of M r = 140000 and 1085 amino acid residues per enzyme molecule) [3]. The presence of a single negative CD extremum at this wavelength indicates that, like ceruloplasmin [6], Ascorbate Oxidase exists predominantly in the β conformation, similar to that observed in the β form of poly(L-lysine).

Inhibitor binding studies on Ascorbate Oxidase

Abstract The characteristic features of the plant multicopper enzyme Ascorbate Oxidase are described, together with the current knowledge about its catalytic mechanism and substrate specificity. A variety of small anionic inhibitors have been used as spectroscopic probes for the enzyme metal sites, but recently interest has arisen for a new type of phenolic inhibitors which act competitively against Ascorbate. These simple phenolic compounds can bind to the enzyme in the same pocket near type 1 copper as the substrate Ascorbate binds, as shown by docking and molecular mechanics computations.

Inhibition of Ascorbate Oxidase by phenolic compounds. Enzymatic and spectroscopic studies.

Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by Ascorbate Oxidase was investigated at pH 7.0 and 23.0 °C. Inhibition of p-nitrophenol is pH dependent over the range 5.0−8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of Ascorbate Oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (Ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of Ascorbate Oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a cop…