Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.     

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.     

Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera of healthy individuals. Full-length tNOX mRNA is present in both normal and tumor cells but appears not to be expressed in either. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. Four splice variants were found. Of these, the exon 4 minus and exon 5 minus forms present in cancer cell lines were absent in noncancer cell lines. In contrast to full-length tNOX cDNA, transfection of COS cells with tNOX exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the plasma membrane. The exon 4 minus form resulted in initiation of translation at a downstream M231 initiation site distinct from that of full-length mRNA. With replacement of M231 by site-directed mutagenesis, no translation of exon 4 minus cDNA or cell surface expression of 34 kDa mature tNOX was observed. The unprocessed molecular mass of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to that of the principal native tNOX form of the endoplasmic reticulum. Taken together, the molecular basis of cancer-cell-specific expression of 34 kDa tNOX appears to reside in the cancer-specific expression of exon 4 minus splice variant mRNA.     

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.     

The plasma membrane NADH oxidase of soybean has vitamin K(1) hydroquinone oxidase activity

Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K(1) hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K(1) (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K(1) reduction or oxygen consumption. The K(m) for reduced K(1) was 350 microM. With the partially purified enzyme, reduced vitamin K(1) was oxidized at a rate of about 600 nmol/min/mg protein and the K(m) was 400 microM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 microM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2, 4-dichlorophenoxyacetic acid. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via reduced vitamin K(1) to acceptors (molecular oxygen or protein disulfides) at the cell surface.     

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity.

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.     

Stress-induced down-regulation of tumor-associated NADH oxidase during apoptosis in transformed cells

Tumor-associated NADH oxidase (tNOX) is a growth-related protein expressed in transformed cells. tNOX knockdown using RNA interference leads to a significant reduction in HeLa cell proliferation and migration, indicating an important role for tNOX in growth regulation and the cancer phenotype. Here, we show that tNOX is down-regulated during apoptosis in HCT116 cells. Treatment with diverse stresses induced a dose- and time-dependent decrease in tNOX expression that was concurrent with apoptosis. Moreover, shRNA-mediated tNOX knockdown rendered cells susceptible to apoptosis, whereas re-expression of tNOX partially recovered cell proliferation. Our results indicate that tNOX is suppressed during apoptosis and demonstrate that tNOX down-regulation sensitizes cells to stress-induced growth reduction, suggesting that tNOX is required for transformed cell growth.     

An aging-related cell surface NADH oxidase (arNOX) generates superoxide and is inhibited by coenzyme Q

This report describes a novel ECTO-NOX protein with an oscillating activity having a period length of ca. 26 min encountered with buffy coat fractions and sera of aged individuals (70–100 years) that generates superoxide as measured by the reduction of ferricytochrome c. The oscillating, age-related reduction of ferricytochrome c is sensitive to superoxide dismutase, is inhibited by coenzyme Q and is reduced or absent from sera of younger individuals (20–40 years). An oscillating activity with a regular period length is a defining characteristic of ECTO-NOX proteins (a group of cell surface oxidases with enzymatic activities that oscillate). The period length of ca. 26 min is longer than the period length of 24 min for the usual constitutive (CNOX) ECTO-NOX proteins of the cell surface and sera which neither generate superoxide nor reduce ferricytochrome c. The aging-related ECTO-NOX protein (arNOX) provides a mechanism to transmit cell surface oxidative changes to surrounding cells and circulating lipoproteins potentially important to atherogenesis. Additionally, the findings provide a rational basis for the use of dietary coenzyme Q to retard aging-related arterial lesions.     

hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2)

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.     

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.     

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 microM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 microM adriamycin with 1 and 10 microM adriamycin being less inhibitory than 0.1 microM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.     

Essential role of copper in the activity and regular periodicity of a recombinant,tumor-associated,cell surface,growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2)

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.     

Catalase-peroxidases (KatG) exhibit NADH oxidase activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.     

Down-Regulation of Tumor-Associated NADH Oxidase,tNOX (ENOX2), Enhances Capsaicin-Induced Inhibition of Gastric Cancer Cell Growth

Gastric cancer is a common human malignancy and a major contributor to cancer-related deaths worldwide. Unfortunately, the prognosis of most gastric cancer patients is poor because they are generally diagnosed at a late stage after the cancer has already metastasized. Most current research, therefore, emphasizes selective targeting of cancer cells by apoptosis-inducing agents. One such therapeutic agent is capsaicin, a component of chili peppers that has been shown to possess anti-growth activity against various cancer cell lines. Here, we examined the effect of capsaicin on SNU-1 and TMC-1 gastric cancer cells and found differing outcomes between the two cell lines. Our results show that capsaicin induced significant cytotoxicity with increases in oxidative stress, PARP cleavage, and apoptosis in sensitive SNU-1 cells. In contrast, TMC-1 cells were much less sensitive to capsaicin, exhibiting low cytotoxicity and very little apoptosis in response to capsaicin treatment. Capsaicin-induced apoptosis in SNU-1 cells was associated with down-regulation of tumor-associated NADH oxidase (tNOX) mRNA and protein. On the contrary, tNOX expression was scarcely affected by capsaicin in TMC-1 cells. We further showed that tNOX-knockdown sensitized TMC-1 cells to capsaicin-induced apoptosis and G1 phase accumulation, and led to decreased cell growth, demonstrating that tNOX is essential for cancer cell growth. Collectively, these results indicate that capsaicin induces divergent effects of the growth of gastric cancer cells that parallel its effects on tNOX expression, and demonstrate that forced tNOX down-regulation restored capsaicin-induced growth inhibition in TMC-1 cells.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

Aqueous two-phase partition and detergent precipitation of a drug-responsive NADH oxidase from the HeLa cell surface

The partitioning behaviour of a drug (capsaicin)-responsive NADH oxidase (tNOX) activity released from HeLa ceIls by low pH treatment followed by heat and proteinase K was determined. When partitioned in a standard 6.4% PEG 3350/6.4% dextran T-500 two-phase system, the bulk of the tNOX activity was in the dextran-rich lower phase. The activity was inhibited by and bound to the triazine dye, Cibacron blue. Affinity partition, where the Cibacron blue was coupled to amino PEG 5000 and added to the first two-phase separation step, resulted in the partitioning of activity to the upper PEG phase. A second partition with PEG-salts resulted in the release of the tNOX from the Cibacron blue–amino PEG enriched phase into the salt-enriched lower phase. The phase-purified protein exhibited anomalous behavior and tended to multimerize in sodium dodecyl sulphate (SDS) prior to SDS-polyacrylamide gel electrophoresis (PAGE). Multimerization appeared to be enhanced by PEG. The multimerization was enhanced with the reduced protein in the presence of detergent prior to SDS–PAGE. In addition, the activity was precipitated by PEG 8000 at concentrations between 6 and 30% by weight. In the presence of or after exposure to PEG 3350 or PEG 8000, the protein could not be detected by Western blot analysis after SDS–PAGE suggesting that the protein failed to enter the gel even though other HeLa cell surface proteins were unaffected. The anomalous multimerization behavior has thus far precluded the use of phase partition as a practical purification step for the oxidase.     

Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae.

NADH oxidase (EC 1.6.99.3) was purified from cell lysates of Serpulina (Treponema) hyodysenteriae B204 by differential ultracentrifugation, ammonium sulfate precipitation, and chromatography on anion-exchange, dye-ligand-affinity, and size-exclusion columns. Purified NADH oxidase had a specific activity 119-fold higher than that of cell lysates and migrated as a single band during denaturing gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]). The enzyme was a monomeric protein with an estimated molecular mass of 47 to 48 kDa, as determined by SDS-PAGE and size-exclusion chromatography. Optimum enzyme activity occurred in buffers with a pH between 5.5 and 7.0. In the presence of oxygen, beta-NADH but not alpha-NADH, alpha-NADPH, or beta-NADPH was rapidly oxidized by the enzyme (Km = 10 microM beta-NADH; Vmax = 110 mumol beta-NADH min-1 mg of protein-1). Oxygen was the only identified electron acceptor for the enzyme. On isoelectric focusing gels, the enzyme separated into three subforms, with isoelectric pH values of 5.25, 5.35, and 5.45. Purified NADH oxidase had a typical flavoprotein absorption spectrum, with peak absorbances at wavelengths of 274, 376, and 448 nm. Flavin adenine dinucleotide was identified as a cofactor and was noncovalently associated with the enzyme at a molar ratio of 1:1. Assays of the enzyme after various chemical treatments indicated that a flavin cofactor and a sulfhydryl group(s), but not a metal cofactor, were essential for activity. Hydrogen peroxide and superoxide were not yielded in significant amounts by the S. hyodysenteriae NADH oxidase, indirect evidence that the enzyme produces water from reduction of oxygen with NADH. The N-terminal amino acid sequence of the NADH oxidase was determined to be MKVIVIGCHGAGTWAAK. In its biochemical properties, the NADH oxidase of S. hyodysenteriae resembles the NADH oxidase of another intestinal bacterium, Enterococcus faecalis.     

Purification and properties of NADH oxidase from Bacillus megaterium

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5'-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr = 52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KPi buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.