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• W2094374311 abstract "l-Galactono-γ-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of l-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%. The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography. The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent K m of 3.3 mm forl-galactono-γ-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated. The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da. Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted withl-galactono-γ-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of l-ascorbic acid biosynthesis in vivo. l-Galactono-γ-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of l-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%. The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography. The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent K m of 3.3 mm forl-galactono-γ-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated. The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da. Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted withl-galactono-γ-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of l-ascorbic acid biosynthesis in vivo. Vitamin C or ascorbic acid (l-AA) 1The abbreviations used are: l-AA,l-ascorbic acid; FPLC, fast protein liquid chromatography; HPLC, high-performance liquid chromatography; l-GL,l-galactono-γ-lactone; l-GuL,l-gulono-γ-lactone; GLDase,l-galactono-γ-lactone dehydrogenase; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s). is an important metabolite for most living organisms present in millimolar concentrations and is well known for its antioxidant properties. Its precise functions in plants is still poorly understood, although it is known to play an important role in the antioxidant system that protects plants from oxidative damage resulting from biotic and abiotic stresses as well as being a cofactor for a number of hydroxylase enzymes. l-AA is synthesized by all higher plants and by nearly all higher animals except humans, other primates, guinea pigs, bats, and some birds (1Burns J.J. Nature. 1957; 180: 533Crossref Scopus (0) Google Scholar, 2Chaudhuri C.R. Chatterjee I.B. Science. 1969; 164: 435-436Crossref PubMed Scopus (62) Google Scholar, 3Chatterjee I.B. Science. 1973; 182: 1271-1272Crossref PubMed Scopus (334) Google Scholar). l-AA has also been reported to be present in a number of yeasts (4Heick H.M.C. Graff G.L.A. Humpers J.E.C. Can. J. Microbiol. 1972; 18: 597-600Crossref PubMed Scopus (33) Google Scholar), but several reports suggest thatl-AA analogues, rather than l-AA, are present in microorganisms (5Takahashi T. Yamashita H. Kato E. Mitshumoto M. Murakawa S. Agric. Biol. Chem. 1976; 40: 121-129Google Scholar, 6Leung C.T. Loewus F.A. Plant Sci. 1985; 38: 65-69Crossref Scopus (17) Google Scholar, 7Nick J.A. Leung C.T. Loewus F.A. Plant Sci. 1986; 46: 181-187Crossref Scopus (39) Google Scholar). The biosynthesis of l-AA follows different pathways in the animal and the plant kingdom. In animals, d-glucose serves as the first committed precursor in the biosynthesis ofl-AA and the last step in the pathway is catalyzed by a microsomal l-gulono-γ-lactone oxidase (EC 1.1.3.8), which oxidizes l-gulono-γ-lactone (l-GuL) tol-AA. This enzyme has been isolated and characterized from rat, goat, and chicken (8Nishikimi M. Tolbert B. Udenfriend S. Arch. Biochem. Biophys. 1976; 175: 427-435Crossref PubMed Scopus (59) Google Scholar, 9Kiuchi K. Nishikimi M. Yagi K. Biochemistry. 1982; 21: 5076-5082Crossref PubMed Scopus (66) Google Scholar). Despite the importance of l-AA in plants, the biosynthetic pathway has still not been established, although current evidence suggests the existence of two discrete routes. A biosynthetic pathway from d-galactose proceeding vial-galactono-γ-lactone (l-GL) has been proposed as long ago as 1954 by Isherwood et al. (10Isherwood F.A. Chen Y.T. Mapson L.W. Biochem J. 1954; 56: 1-15Crossref PubMed Scopus (76) Google Scholar) and Mapson et al. (11Mapson L.W. Isherwood F.A. Chen Y.T. Biochem. J. 1954; 56: 21-28Crossref PubMed Scopus (40) Google Scholar), based on initial studies of the oxidation of l-GL to l-AA by the enzymel-galactono-γ-lactone dehydrogenase (GLDase). GLDase activity has been described (11Mapson L.W. Isherwood F.A. Chen Y.T. Biochem. J. 1954; 56: 21-28Crossref PubMed Scopus (40) Google Scholar, 12Mapson L.W. Breslow E. Biochem. J. 1958; 68: 395-406Crossref PubMed Scopus (76) Google Scholar, 13Ôba K. Fukui M. Imai Y. Iriyama S. Nogami K. Plant Cell Physiol. 1994; 35: 473-478Google Scholar) in plants such as pea, cabbage, cauliflower florets, and potato, and recently Ôba et al. (14Ôba K. Ishikawa S. Nishikawa M. Mizuno H. Yamamoto T. J. Biochem. (Tokyo). 1995; 117: 120-124Crossref PubMed Scopus (133) Google Scholar) reported a purification of this enzyme from sweet potato roots. Loewus (15Loewus F.A. The Biochemistry of Plants. 14. Academic Press, New York1988: 85-107Google Scholar) has proposed an alternative pathway in whichl-AA is synthesized from d-glucose vial-sorbosone. The presence of an enzyme able to convertl-sorbosone to l-AA with concomitant reduction of NADP was demonstrated in bean and spinach leaves (16Loewus M.W. Bedgar D.L. Saito K. Loewus F.A. Plant Physiol. 1990; 94: 1492-1495Crossref PubMed Scopus (47) Google Scholar, 17Saito K. Nick J.A. Loewus F.A. Plant Physiol. 1990; 94: 1496-1500Crossref PubMed Scopus (43) Google Scholar). Conceivably, these distinct routes might be present in different subcellular compartments or in different plant species. Here, we report the purification and characterization of GLDase from cauliflower florets, followed by isolation and sequencing of the corresponding cDNA. This is the first description of a gene coding for an enzyme involved in the biosynthesis of l-AA in plants. The GLDase cDNA has furthermore been expressed in an active form in yeast, and we have strong indications that the substrate for GLDase, l-GL is naturally present in plant extracts. These findings emphasize for the first time the physiological relevance of the biosynthetic pathway proposed by Isherwood et al. and Mapson et al. (10Isherwood F.A. Chen Y.T. Mapson L.W. Biochem J. 1954; 56: 1-15Crossref PubMed Scopus (76) Google Scholar, 11Mapson L.W. Isherwood F.A. Chen Y.T. Biochem. J. 1954; 56: 21-28Crossref PubMed Scopus (40) Google Scholar). Sephacryl SF-200, DEAE-Sepharose, and phenyl-Sepharose CL-4B were obtained from Pharmacia (Uppsala, Sweden).l-Galactono-γ-lactone,d-galactono-γ-lactone, d-gulono-γ-lactone,l-gulono-γ-lactone, l-mannono-γ-lactone,d-galactonic acid, d-glucuronic acid,d-gluconic acid, and p-hydroxymercuribenzoic acid were from Sigma. d-Erythronic-γ-lactone,d-xylonic-γ-lactone, and N-ethylmaleimide were purchased from Aldrich. Restriction enzymes were from Pharmacia and [α-32P]dCTP was from Amersham (Aylesbury, United Kingdom). Cauliflowers (Brassica oleracea, var. botrytis) were obtained from a field nearby Gent and kept at 4 °C until use. Cauliflower florets (7.5 kg) were cut into small pieces and homogenized in a pre-cooled blender in ice-cold buffer A (400 mm sucrose, 100 mm sodium phosphate buffer, pH 7.4) at 1 liter/kg fresh weight. The homogenate was passed through four layers of Miracloth tissue (Calbiochem-Novabiochem, La Jolla, CA), and centrifuged at 13,500 × g for 45 min in a GS3 rotor. The pellet containing the mitochondria (approximately 250 g of material) was stored at −70 °C until further use. The crude, frozen mitochondrial pellet was gently thawed in a microwave oven and resuspended in 1/10 volume (750 ml) of buffer A. Cold acetone (−20 °C) was slowly added while stirring (10 × volumes) and the mixture was allowed to stand for 30 min at 4 °C. Precipitated proteins were then collected by filtration through pre-filter paper (A15; Millipore, Bedford, MA) and resuspended in 1/10 volume of buffer B (40 mm Tris-HCl, pH 9.0) followed by 5 h dialysis against 10 volumes of buffer B. The denatured proteins were removed by centrifugation (10,000 × g for 15 min). GLDase was then purified from the supernatant and designated as the protein extract, using the protocol described below (“Enzyme Purification”). All manipulations concerning the preparation of extracts and enzyme purification were carried out at 4 °C, unless stated otherwise. GLDase activity was measured spectrophotometrically by following thel-GL-dependent reduction of cytochromec at 550 nm and 22 °C. The reaction mixture (1 ml) consisted of enzyme extract, cytochrome c (1.5 mg/ml), andl-GL (4.2 mm) in 0.05 m Tris-HCl buffer (pH 8.4). Under these conditions the reaction rate was linear with respect to time for an initial period of at least 15 min. One unit of enzyme activity was defined as the amount that oxidized 1 μmol ofl-AA/min. This corresponds to the reduction of 2 μmol of cytochrome c as described by Ôba et al.(13Ôba K. Fukui M. Imai Y. Iriyama S. Nogami K. Plant Cell Physiol. 1994; 35: 473-478Google Scholar). Substrate specificity assays were carried out as described above using 4.2 mm of the different substrates to be tested. The protein extract (from 250 g of mitochondrial pellet) was loaded onto a DEAE-Sepharose column (5 × 12 cm) equilibrated with buffer B. After washing with 4 column volumes of buffer B at 60 ml/h, elution was carried out with 0.5m NaCl in the same buffer. Fractions of 8 ml were collected at a flow rate of 60 ml/h, and fractions containing GLDase activity were pooled and ammonium sulfate was added to a concentration of 1m. The extract was then loaded on a phenyl-Sepharose CL-4B column (2.2 × 15.0 cm) equilibrated in buffer C (1 mammonium sulfate, 25 mm sodium phosphate, pH 7.0). After washing with 2 column volumes of buffer C, elution was carried out at 30 ml/h by mixing buffer C with a 600-min linear gradient of 80% ethylene glycol in 25 mm sodium phosphate (pH 7.0). Fractions containing GLDase activity were again pooled, concentrated to 10 ml by ultrafiltration using a PM-10 membrane (Amicon, Beverly, MA), and then applied onto a Sephacryl SF-200 gel filtration column (2.6 × 94 cm) equilibrated in buffer D (20% ethylene glycol, 40 mm NaCl, 80 mm sodium phosphate, pH 7.4). The enzyme was eluted with the same buffer at a flow rate of 25 ml/h. Fractions of 5 ml were collected and fractions with activity pooled. This preparation could be stored at 4 °C for several weeks without any detectable loss of activity. Two gel filtration preparations were combined and concentrated with buffer exchange to buffer E (20% ethylene glycol, 20 mmTris-HCl, pH 8.0) by ultrafiltration (PM-10 membrane). The resulting solution was applied to a strong anion exchange column (Resource Q, 6 ml; Pharmacia Biotech Inc.) equilibrated in buffer E and connected to an FPLC system (Pharmacia). The column was eluted at 1 ml/min with a gradient of 0–450 mm NaCl in buffer E as follows: 0–85 mm in 18 min, 85–110 mm in 10 min, 110–130 mm in 14 min, and 130–450 mm in 10 min. Fractions of 1 ml were collected. The activity of the main peak, which eluted at 120 mm NaCl, was collected and adjusted to pH 6.0 with 50 mm sodium phosphate. The pooled fractions were loaded onto a Poros 20 SP strong cation exchange column (PerSeptive Biosystems, Cambridge, MA) equilibrated in buffer F (20 mm sodium phosphate, pH 6.0, 20% ethylene glycol) and eluted using the FPLC at a flow rate of 1 ml/min. Elution was carried out with a gradient of 0–500 mm NaCl in buffer F as follows: 125–225 mm in 40 min and 225–500 mm in 37 min. Fractions of 2 ml were collected. Two peaks of activity eluted: peak I at 210 mm and peak II at 225 mm NaCl. Peak II was dialyzed against 10 mmsodium phosphate, pH 7.2, containing 1 mml-AA and the volume was reduced to 200 μl by lyophilization (Heto Lab Equipment, Lyngby, Denmark). As a final step, the pooled fractions of peak II were separated by HPLC using a Zorbax gel filtration column GF-250 (9.4 × 250 mm) (Rockland Technologies Inc., Newport, DE) equilibrated in 750 mm NaCl, 50 mm sodium phosphate (pH 7.2). Fractions of 1 ml were collected at a flow rate of 1 ml/min. The protein concentration of extracts was determined according to Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as standard. The molecular mass of the native GLDase was estimated by gel filtration on a Sephacryl SF-200 column (2.5 × 94 cm) equilibrated in 40 mm NaCl, 80 mm sodium phosphate (pH 7.4). The column was eluted at a flow rate of 20 ml/h and fractions of 4 ml were collected. The molecular mass was estimated by comparing the elution of GLDase with that of the standard proteins: ferritin (450 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.5 kDa). Analytical SDS-PAGE was performed in slab gels of 10% polyacrylamide as according to Chua (19Chua N.H. Methods Enzymol. 1980; 69: 434-446Crossref Scopus (331) Google Scholar). Proteins were visualized either by Coomassie Brilliant Blue R-250 staining (19Chua N.H. Methods Enzymol. 1980; 69: 434-446Crossref Scopus (331) Google Scholar) or silver nitrate staining (20Merril C.R. Goldman D. Van Keuren M.L. Methods Enzymol. 1984; 104: 441-447Crossref PubMed Scopus (282) Google Scholar). Cytochrome cwas covalently bound to thiol-activated Sepharose 4B as described by Azzi et al. (21Azzi A. Bill K. Broger C. Casey R.P. Riess V. Azzi A. Brodbeck U. Zahler P. Membrane Proteins: A Laboratory Manual. Springer Verlag, Berlin1981Crossref Google Scholar) and packed into a column (1.0 × 20 cm) that eluted at flow rates of 8 ml/h in 10 mm sodium phosphate buffer (pH 7.4). Fractions of 2 ml were collected and tested for activity. Lycorine was purified from non-flowering, whole plants of Crinum jagus or Crinum asiaticum as described by Davey et al. 2M. W. Davey, G. Persiau, A. De Bruyn, J. Van Damme, G. Bauw, and M. Van Montagu, submitted for publication. . Purified GLDase from the Poros 20 SP purification step was applied to SDS-PAGE. The separated polypeptides were blotted onto polyvinylidene difluoride membranes (Millipore) as described by Bauw et al. (23Bauw G. De Loose M. Inzé D. Van Montagu M. Vandekerckhove J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4806-4810Crossref PubMed Google Scholar). NH2-terminal and internal amino acid sequence analyses of the polyvinylidene difluoride-bound proteins were performed as described by Bauw et al. (24Bauw G. Van Damme J. Puype M. Vandekerckhove J. Gesser B. Ratz G.P. Lauridsen J.B. Celis J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7701-7705Crossref PubMed Scopus (149) Google Scholar). Trypsin was used for thein situ digests and the resulting peptides were separated by reversed-phase HPLC. Amino acid sequencing was performed on a 473 protein sequencer (Applied Biosystems, Foster City, CA). Cauliflower floret tissue (300 mg) was ground to a powder in liquid nitrogen with a mortar and pestle and RNA was extracted using a method based on LiCl precipitation as described by Goormachtig et al. (25Goormachtig S. Valerio-Lepiniec M. Szczyglowski K. Van Montagu M. Holsters M. De Bruijn F. Mol. Plant-Microbe Interact. 1995; 8: 816-824Crossref PubMed Scopus (73) Google Scholar). The RNA isolated from cauliflower florets (4 μg) was used to synthesize first-strand cDNA according to the instruction manual for SuperscriptTMPreamplification System for first-strand cDNA synthesis (Life Technologies, Inc., Gaithersburg, MD). Degenerate oligonucleotides were synthesized on an oligonucleotide synthesizer (Applied Biosystems) and used as primers in polymerase chain reactions. The peptide sequences used for synthesizing the corresponding coding and complementary oligonucleotides were designed according to the partial amino acid sequence obtained earlier, and designated 3, 6, and 8 (underlined in Fig. 5). First-strand cDNA synthesized from cauliflower florets was used as a template. The amplification mixture consisted of template, polymerase chain reaction buffer, 200–300 ng of each primer, 2.5 mmcNTP, and 1 unit of Taq polymerase in a total volume of 50 μl. The amplification program consisted of 32 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 1 min), and primer extension (72 °C, 2 min). Products of the reaction were separated on 1% agarose gels, excised, and then purified according to the QIAEX Handbook (Diagen GmbH, Hilden, Germany). The purified products were cloned into a pGEM-T vector (Promega, Madison, WI). A cauliflower cDNA library constructed in λZAP II (Stratagene, La Jolla, CA) was used. Aliquots of the cDNA library were plated out usingEscherichia coli XL-1 Blue cells on 23 × 23-cm baking plates (Nunc, Roskilde, Denmark) containing NZY agar. Approximately 600,000 plaques of the library were transferred onto duplicate nylon membranes (Hybond N+; Amersham). The membranes were treated in accordance with the manufacturer's instructions for plaque blotting. DNA was fixed to membranes by irradiation with ultraviolet light (UV Stratalinker; Stratagene). A 250-bp polymerase chain reaction-amplified fragment was labeled with [α-32P]dCTP using a random primed DNA labeling kit (Boehringer, Mannheim, Germany) and subsequently used as probe for screening the cDNA library. The membranes were washed for 4 h at 65 °C in hybridization buffer (1% (w/v) bovine serum albumin, 7% (w/v) SDS, 1 mm EDTA, and 0.25 m sodium phosphate, pH 7.2), before 20 h incubation with the32P-labeled probe in hybridization buffer at 65 °C. The membranes were then rinsed twice for 15 min with 2 × SSC (1 × SSC: 150 mm NaCl, 15 mmNa3-citrate, pH 7.0) and 1% SDS at room temperature and exposed to X-Omat AR film (Kodak, Rochester, NY) with an enhancer screen for autoradiography. Plaque-purified phage clones were converted into phagemids (Bluescript SK-/+; Stratagene) by in vivoexcision using the ExAssistTM System. DNA sequence determinations were carried out in accordance with protocols obtained from Applied Biosystems. Initial sequences were obtained by use of T7 and T3 vector primers. To complete the sequences on both strands, cDNA-specific primers were used. The sequence analyses were carried out using software of the Genetics Computer Group (Madison, WI). To express the GLDase cDNA in yeast (Saccharomyces cerevisiae), the Bluescript vector containing the full-length cDNA was digested with ApaI andKpnI and a 27-bp adapter containing a NotI restriction site subsequently ligated into theApaI-KpnI-linearized vector. The resulting construct containing two NotI restriction sites was cloned into the NotI restriction sites of the pFL61 vector (26Minet M. Dufour M.E. Lacroute F. Plant J. 1992; 2: 417-422PubMed Google Scholar). Yeast cells of the strain W303B (Matα, ade2, ura3, his3, trp1, leu2, can1-100) (27Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (388) Google Scholar) were transformed by the method of Dohmenet al. (28Dohmen R.J. Strasser A.W.M. Höner C.B. Hollenberg C.P. Yeast. 1991; 7: 691-692Crossref PubMed Scopus (319) Google Scholar) and plated on selective 1.5% agar plates (lacking uracil) containing minimal SD medium (0.2% yeast nitrogen base (Difco, Detroit, MI), 0.7% ammonium sulfate, 2.7% glucose) supplemented with adenine, tryptophan, leucine at 20 μg/ml, and histidine at 10 μg/ml (as above minus agar). Transformed cells were transferred to liquid SD medium and grown for 3 days at 30 °C. The cells were collected by centrifugation (8,000 × g, 15 min), washed, and resuspended in 50 mm Tris-HCl (pH 8.0). For GLDase activity tests and protein determinations the cells were disrupted by two passages through a French Press after a cycle of freezing (−70 °C) and thawing. Up to 1 g of plant tissue was first thoroughly homogenized using a pestle and mortar in liquid nitrogen, and extracted using 10% trichloroacetic acid to precipitate proteins and inhibit degradative enzymes. After filtration and partitioning against water-saturated diethyl ether to remove trichloroacetic acid, samples were concentrated and injected onto the C18 HPLC column, eluted with 0.1% trifluoroacetic acid. Peaks eluting in the region of l-GL elution were collected and tested for their ability to serve as a substrate for GLDase. Analogous peaks from up to 10 runs were combined, dried under vacuum, and re-injected onto the aminopropyl HPLC column for weak anion exchange. Once again peaks eluting in the region of thel-GL standard were collected and tested for their ability to serve as a substrate for GLDase. Positive peaks from several runs were pooled, concentrated, and finally reinjected on a C18 reversed-phase HPLC column eluted with phosphoric acid (pH 2.5). HPLC was carried out using a 600E pump (Waters, Milford, MA) and a Waters 996 diode-array detector. Injections (20–40 μl) were made using a WISP 412 (Waters) autosampler onto a C18, 3-μm spherical particle size, 250 × 4.6 mm inner diameter, reversed-phase HPLC column (Bio-Rad), fitted with a 10-mm guard column. Separations were carried out isocratically at 800 μl/min with phosphoric acid (pH 2.5), or 0.1% trifluoroacetic acid as mobile phase. Data were collected and analyzed, and the entire system was controlled using the Millenium 2010 (v1.15) chromatography management system (Waters). Weak anion exchange separations were carried on a 250 × 3.6-mm aminopropyl column (Phenomenex Inc., Torrance, CA), eluted isocratically with 15% (v/v) 20 mmKH2PO4 (pH 6.0), in acetonitrile. The column was regenerated after each analysis with a 10-min linear gradient of 15–50% acetonitrile in 20 mmKH2PO4 (pH 6.0) at 1 ml/min. Strong anion exchange HPLC with pulsed amperometric electrochemical detection was carried out on the same system fitted with an HP 1049A electrochemical detector containing a gold amalgam-working electrode at an operating potential of +100 mV. Separations were performed on a 300 × 4.6-mm, Dionex PA-100 strong anion exchange column (Dionex Corp., Sunnyvale, CA) eluted with a 20-min linear gradient of 0 to 200 mm sodium acetate in 3 ml/liter NaOH. A summary of the purification of GLDase from cauliflower florets is presented in Table I. As the enzymatic activity was found to be most stable in 20% ethylene glycol this reagent was included in all buffers except for buffers A and B used in the first two purification steps. Interestingly, after the DEAE-Sepharose step the total GLDase activity increased slightly, probably due to removal of inhibitory compounds present in the crude extract. The first three purification steps had relatively little influence on the purity of GLDase, but the FPLC Resource step (strong anion exchange) resulted in an increase in the purification factor from 63 to 900, although there was a corresponding decrease in recoveries to only 47% compared with the activity present in the gel filtration pool. After passage through the strong cation exchange column (Poros 20 SP), GLDase activity was resolved into two peaks designated I and II (Fig. 1). The activity forming the latter peak was used for further analysis. At this stage GLDase was purified 1693-fold from the initial mitochondrial fraction with a recovery of 1.1% (Table I). The purity of the final enzyme preparation was confirmed by SDS-PAGE, where we consistently obtained three polypeptide bands corresponding to approximately 56, 30, and 26 kDa (Fig.2). Further purification of the enzyme by a high resolution gel filtration on a Zorbax GF 250 column did not result in elimination of the 30- and 26-kDa polypeptide bands; and subsequent amino acid sequence analyses revealed them to be breakdown products of the 56-kDa band. The native molecular mass of the enzyme was estimated to be approximately 56 kDa by Sephacryl SF-200 (Fig.3) and Zorbax GF 250 high resolution gel filtration.Table IPurification scheme for GLDaseStepVolumeProteinActivityFoldRecoveryTotalSpecificmlmgunitsunits/mg%Acetone precipitation2,5001,51044,900301100DEAE ion exchange835546,50084528104Phenyl-Sepharose382130,8001,4674969Gel filtration541120,9001,9006347FPLC Resource Q320.38,10027,00090018FPLC Poros 20 SP40.0150850,8001,6931.1Mitochondrial extract from 15 kg of cauliflower florets were used for the preparation Open table in a new tab Figure 2SDS-PAGE. Lane A, molecular mass standards; lane B, GLDase peak II from the Poros SP (strong anion exchange) purification step, analyzed by SDS-PAGE after an additional high-resolution HPLC gel filtration step. A polypeptide band corresponding to approximately 56 kDa (GLDase), and two degradation products of 30 and 26 kDa (confirmed by amino acid sequence analyses) were visualized by silver nitrate staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Estimation of molecular mass of GLDase.The native molecular mass was estimated by gel filtration chromatography on Sephacryl SF-200. The arrow indicates GLDase activity. Molecular mass standards used were: 1) ferritin (450 kDa); 2) alcohol dehydrogenase (150 kDa);3) bovine serum albumin (66 kDa); 4) carbonic anhydrase (29 kDa); and 5) cytochrome c (12.5 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mitochondrial extract from 15 kg of cauliflower florets were used for the preparation NH2-terminal sequence analysis of the complete 56- and 30-kDa polypeptide bands were found to be identical, and the partially determined sequence of the 26-kDa band was located within the deduced amino acid sequence of the GLDase cDNA (Asp-273 to Leu-289). Trypsin digestions of the 56-kDa protein yielded a series of peptides which were separated by reversed-phase HPLC. A number of the peptides were subjected to partial sequence analysis and could again be located in the GLDase cDNA, as indicated in Fig. 5. Various isomeric compounds were tested as possible substrates for the purified GLDase using cytochrome c as electron acceptor. These werel-GL, d-galactono-γ-lactone,d-gulono-γ-lactone, l-GuL,d-erythronic-γ-lactone,d-xylonic-γ-lactone, l-mannono-γ-lactone,d-galactonic acid, d-glucuronic acid, andd-gluconic acid. Apart from l-GL, none of the compounds tested could serve as a substrate for GLDase because no reduction of cytochrome c was observed. GLDase obeyed Michaelis-Menten-type kinetics using l-GL as substrate. With the method of Lineweaver and Burk (Fig.4), the K m value was determined to be 3.3 mm with a V maxof 7.1 units/min. Concentrations of l-GL used were from 1.0 to 32.6 mm. Substrate inhibition was observed at 32.6 mm. The pH dependence of the enzyme activity was examined using 50 mm sodium phosphate buffer in the pH range from 6.0 to 7.6 and 50 and 100 mm Tris-HCl in the range between 7.4 and 8.8 at 22 °C with 4.2 mml-GL. A broad maximum of activity between pH 8.0 and 8.5 was observed (results not shown). The enzyme assay is based on the reduction of cytochrome c by GLDase, in which for each micromole of oxidized l-GL, 2 μmol of cytochrome c are reduced, because the l-AA formed is spontaneously oxidized by cytochrome c to dehydroascorbic acid. The purified GLDase showed strict specificity for cytochrome c, and neither FAD, NAD, NADP, nor molecular oxygen were able to serve as electron acceptors for the enzyme. The effect of various substrate analogues, organic inhibitors, and some divalent metal ions were examined for their influence on the enzyme activity. The oxidation ofl-GL by GLDase was tested in the presence of equimolar concentrations of each of the following compounds:d-galactono-γ-lactone, d-gulono-γ-lactone,l-gulono-γ-lactone, d-erythronic-γ-lactone,d-xylonic-γ-lactone, l-mannono-γ-lactone,d-galactonic acid, d-glucuronic acid, andd-gluconic acid. None of these had any influence on the reaction rate. Of the divalent metal salts we tested, MgCl2, CaCl2, and SrCl2 had no effect on the GLDase activity at concentrations up to 15 mm. The chelating agent EDTA had no significant effect on the enzyme activity supporting the conclusion that there was no metal requirement for the enzymatic activity. Sulfhydryl-modifying agents, however, were able to partially" @default.
• W2094374311 created "2016-06-24" @default.
• W2094374311 creator A5006061521 @default.
• W2094374311 creator A5047966980 @default.
• W2094374311 creator A5059345521 @default.
• W2094374311 creator A5069064826 @default.
• W2094374311 creator A5076380781 @default.
• W2094374311 date "1997-11-01" @default.
• W2094374311 modified "2023-10-12" @default.
• W2094374311 title "Isolation of a cDNA Coding forl-Galactono-γ-Lactone Dehydrogenase, an Enzyme involved in the Biosynthesis of Ascorbic Acid in Plants" @default.
• W2094374311 cites W112824158 @default.
• W2094374311 cites W1501604089 @default.
• W2094374311 cites W1573941143 @default.
• W2094374311 cites W1582421052 @default.
• W2094374311 cites W1587794537 @default.
• W2094374311 cites W1611774290 @default.
• W2094374311 cites W1660143940 @default.
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• W2094374311 cites W1983535701 @default.
• W2094374311 cites W1991760647 @default.
• W2094374311 cites W2001139858 @default.
• W2094374311 cites W2004142323 @default.
• W2094374311 cites W2020003263 @default.
• W2094374311 cites W2032306084 @default.
• W2094374311 cites W2035663056 @default.
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• W2094374311 cites W2050499412 @default.
• W2094374311 cites W2051213795 @default.
• W2094374311 cites W2067605088 @default.
• W2094374311 cites W2089020817 @default.
• W2094374311 cites W2090929495 @default.
• W2094374311 cites W2091864583 @default.
• W2094374311 cites W2100717262 @default.
• W2094374311 cites W2141338995 @default.
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• W2094374311 cites W2313896494 @default.
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