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• W2023160617 abstract "Nectarin I, a protein that accumulates in the nectar of Nicotiana sp., was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 °C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H2O2 or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn2+ addition. Addition of Fe2+, Cu2+, Zn2+, and Cu2+/Zn2+ did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H2O2. Putative nectarin I homologues were found in the nectars of several other plant species. Nectarin I, a protein that accumulates in the nectar of Nicotiana sp., was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 °C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H2O2 or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn2+ addition. Addition of Fe2+, Cu2+, Zn2+, and Cu2+/Zn2+ did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H2O2. Putative nectarin I homologues were found in the nectars of several other plant species. germin-like protein superoxide dismutase polyacrylamide gel electrophoresis periodic acid Schiff matrix-assisted laser desorption/ionization time-of-flight 4-morpholinepropanesulfonic acid N,N,N',N'-tetramethylethylenediamine Floral nectars are often considered as being little more than sugar water. However, closer examination reveals a complex mixture of components. Although simple carbohydrates (i.e. sucrose, glucose, and fructose) make up the most significant solutes in nectar, other substances such as amino acids, organic acids, terpenes, flavonoids, glycosides, vitamins, phenolics, oils, and metal ions have also been found in various nectars (1Baker H.G. Baker I. Bentley B. Elias T. The Biology of Nectaries. Columbia University Press, New York1983: 126-152Google Scholar). Enzymatic activities such as invertase, transglucosidase, tyrosinase, phosphatase, oxidase, esterase, and malate dehydrogenase have been suggested to occur in nectars (1Baker H.G. Baker I. Bentley B. Elias T. The Biology of Nectaries. Columbia University Press, New York1983: 126-152Google Scholar). However, these reports have primarily been undetailed investigations, failing to identify the proteins responsible for the respective activities. Only a few investigations have clearly identified the activities of defined nectar proteins (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar, 3Peumans W.J. Smeets K. Van Nerum K. Van Leuven F. Van Damme E.J.M. Planta. 1997; 201: 298-302Crossref PubMed Scopus (61) Google Scholar, 4Scala J. Iott K. Schwab W. Semersky F.E. Plant Physiol. 1969; 44: 367-371Crossref PubMed Google Scholar, 5Heslop-Harrison Y. Knox R.B. Planta. 1971; 96: 183-211Crossref PubMed Scopus (69) Google Scholar). We have previously demonstrated the presence of a limited number of proteins, termed nectarins, that are secreted into the nectar of tobacco flowers (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). The most highly expressed of these proteins, nectarin I, is found only in nectary tissues and to a much lower level in the ovary. Its expression is developmentally regulated, accumulating only at times when nectar is being actively secreted. Following the isolation and characterization of the nectarin I gene, this protein was identified as a germin-like protein (GLP).1 Germin was first identified in germinating wheat embryos (6McCubbin W.C. Cyril M.K. Kennedy T.D. Lane B.G. Biochem. Cell Biol. 1987; 65: 1039-1048Crossref Google Scholar). It is a large molecular-weight protein composed of five (6McCubbin W.C. Cyril M.K. Kennedy T.D. Lane B.G. Biochem. Cell Biol. 1987; 65: 1039-1048Crossref Google Scholar) or six (7Gane P.J. Dunwell J.M. Warwicker J. J. Mol. Evol. 1998; 46: 488-493Crossref PubMed Scopus (61) Google Scholar, 8Woo E.-J. Dunwell J.M. Goodenough P.W. Pickersgill R.W. FEBS Lett. 1998; 437: 87-90Crossref PubMed Scopus (46) Google Scholar) monomer subunits. GLPs have subsequently been identified in all species examined to date from mosses to gymnosperms and dicots to monocots (9Yamahara T. Shiono Y. Suzuki T. Tanaka K. Takio S. Sato K. Yamazaki S. Satoh T. J. Biol. Chem. 1999; 274: 33274-33278Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Domon J.-M. Dumas B. Lainé E. Meyer Y. Alain D. David H. Plant Physiol. 1995; 108: 141-148Crossref PubMed Scopus (67) Google Scholar, 11Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1998; 38: 929-943Crossref PubMed Scopus (60) Google Scholar, 12Rahman S. Grzelczak Z. Kennedy T. Lane B. Biochem. Cell Biol. Biochim. Biol. Cell. 1988; 66: 100-106Crossref PubMed Scopus (8) Google Scholar). Germin is an oxalate oxidase that degrades oxalic acid into H2O2 and CO2 (13Lane B.G. Dunwell J.M. Ray J.A. Schmitt M.R. Cuming A.C. J. Biol. Chem. 1993; 268: 12239-12242Abstract Full Text PDF PubMed Google Scholar, 14Dumas B. Sailland A. Cheviet J.P. Freyssinet G. Pallett K. C. R. Acad. Sci. III. 1993; 316: 793-798PubMed Google Scholar, 15Zhang Z. Collinge D.B. Thordal-Christensen H. Plant J. 1995; 8: 139-145Crossref Scopus (179) Google Scholar). Despite the high sequence identity between nectarin I and germin, nectarin I lacks oxalate oxidase activity (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar) and consequently has an unknown function. Many other GLPs also lack oxalate oxidase activity (9Yamahara T. Shiono Y. Suzuki T. Tanaka K. Takio S. Sato K. Yamazaki S. Satoh T. J. Biol. Chem. 1999; 274: 33274-33278Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10Domon J.-M. Dumas B. Lainé E. Meyer Y. Alain D. David H. Plant Physiol. 1995; 108: 141-148Crossref PubMed Scopus (67) Google Scholar, 16Neutelings G. Domon J. Membre N. Bernier F. Meyer Y. David A. David H. Plant Mol. Biol. 1998; 38: 1179-1190Crossref PubMed Scopus (54) Google Scholar, 17Vallelain-Bindschedler L. Mösinger E. Métraux J.-P. Schweizer P. Plant Mol. Biol. 1998; 37: 297-308Crossref PubMed Scopus (81) Google Scholar, 18Wei Y. Ziguo Z. Andersen C.H. Schmelzer E. Gregersen P.L. Collinge D.B. Smedegaard-Petersen V. Thordal-Christensen H. Plant Mol. Biol. 1998; 36: 101-112Crossref PubMed Scopus (135) Google Scholar, 19Ohmiya A. Tanaka Y. Kadowaki K. Hayashi T. Plant Cell Physiol. 1998; 39: 492-499Crossref PubMed Scopus (58) Google Scholar). Recently, a superoxide dismutase from the moss Barbula unguiculata, BuGLP, was isolated and identified as a GLP (9Yamahara T. Shiono Y. Suzuki T. Tanaka K. Takio S. Sato K. Yamazaki S. Satoh T. J. Biol. Chem. 1999; 274: 33274-33278Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This fortuitous discovery has led us to examine whether the germin-like protein, nectarin I, is also a superoxide dismutase. The plants used for the production of nectarin I have been described previously (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). Additional species examined for the presence of nectarin I are presented in Table I. These plants were obtained from greenhouses on the Iowa State University campus. The species were confirmed at the Iowa State University Herbarium.Table IPurification of nectarin ISOD activity 1-aSuperoxide dismutase activity was evaluated by the method of Flohé and Ötting (24). One unit of activity is defined as the amount of enzyme resulting in 50% inhibition of cytochrome c reduction under standard conditions.Protein 1-bProtein was evaluated by the method of Lowry (27).RecoverySpecific activityPurificationActivityProteinunitsmg%units/mg protein-foldRaw nectar 1-cTo quantitate recovery for this purification, raw nectar was initially dialyzed versus 10 mm sodium phosphate buffer, pH 7.8, to remove materials that interfere with the Lowry protein assay. This dialysis is not required for the purification of nectarin I.1,0481.351001008451.00Heat treatment1,0480.7910063.713771.57(NH4)2SO4precipitation9660.3892.230.625433.01Nectar was harvested as described under “Experimental Procedures.”1-a Superoxide dismutase activity was evaluated by the method of Flohé and Ötting (24Flohé L. Ötting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1311) Google Scholar). One unit of activity is defined as the amount of enzyme resulting in 50% inhibition of cytochrome c reduction under standard conditions.1-b Protein was evaluated by the method of Lowry (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).1-c To quantitate recovery for this purification, raw nectar was initially dialyzed versus 10 mm sodium phosphate buffer, pH 7.8, to remove materials that interfere with the Lowry protein assay. This dialysis is not required for the purification of nectarin I. Open table in a new tab Nectar was harvested as described under “Experimental Procedures.” Several different superoxide dismutases including the MnSOD fromEscherichia coli (20Keele Jr., B. McCord J. Fridovich I. J. Biol. Chem. 1970; 245: 6176-6181Abstract Full Text PDF PubMed Google Scholar), the FeSOD from E. coli(21Yost F.J. Fridovich I. J. Biol. Chem. 1973; 248: 4905-4908Abstract Full Text PDF PubMed Google Scholar), and the Cu/ZnSOD from bovine erythrocytes (22Steinman H. Naik V. Abernethy J. Hill R. J. Biol. Chem. 1974; 249: 7326-7338Abstract Full Text PDF PubMed Google Scholar) were obtained from Sigma and were used without further purification. All other materials were of the highest purity available and were obtained from either Sigma or Fisher. Nectar was collected as described previously (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). The nectarin I protein was obtained in pure form as follows; 12 ml of fresh nectar collected from approximately 500 flowers from 12–15 plants was divided into 600-μl aliquots in 1.5-ml microcentrifuge tubes. The 600-μl aliquots were placed in a 90 °C water bath for 45 min, followed by a 30-min centrifugation at 12,000 × g. To avoid contamination from the pellet, the top 500 μl of nectar was removed, and 10 ml of 100% (NH4)2SO4 was added to each 1.5 ml of nectar (87% final concentration of (NH4)2SO4) and incubated for 1 h in 15-ml Corex tubes. Following incubation, the tubes were centrifuged at 10,000 × g for 15 min. The pellets were resuspended in a minimal volume (150 μl each) of distilled water or 10 mm sodium phosphate, pH 7.8, and dialyzed against 2 L of 10 mm sodium phosphate, pH 7.8, two times. In early studies, partially purified nectar proteins were produced by ammonium sulfate precipitation of raw nectar. Metal ion analysis was performed by flame ionization atomic absorption spectroscopy at the Metal Analysis Laboratory on the Iowa State University campus. All preparations were performed with nitric acid-washed glassware. The procedure described by Sugiura et al. (23Sugiura M. Yamamura H. Haramo K. Sasaki M. Morikava M. Tsuboi M. Chem. Pharm. Bull. 1979; 27: 2003-2007Crossref Scopus (82) Google Scholar) was used for the assay of oxalate oxidase activity in solution, using a commercial preparation of barley oxalate oxidase as a positive control. A colorimetric assay (24Flohé L. Ötting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1311) Google Scholar) using cytochrome c as the detector and xanthine-xanthine oxidase as a superoxide generator was utilized in the characterization of purified nectarin I and in the thermostability studies. SDS-PAGE was performed according to the methods of Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207523) Google Scholar). Western blotting was conducted according to methods of Timmons and Dunbar (26Timmons E.D. Dunbar B.S. Methods Enzymol. 1990; 182: 679-687Crossref PubMed Scopus (114) Google Scholar). Anti-nectarin I antibodies were described previously (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). Protein concentration was determined by the method of Lowry et al. (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Hydrogen peroxide in nectar was evaluated as follows. Fifty microliters of nectar was added to 1.95 ml of distilled water, and then 1 ml of developing solution was added. The developing solution contained 80 μg of 4-aminopyrine, 13 units of horseradish peroxidase and 0.2 μl ofN,N-dimethylanaline in 0.1 m sodium phosphate buffer, pH 5.5. After a 10-min incubation at 37 °C, the absorbance was read at 550 nm. Negative staining of in-gel SOD activity was performed with nitro blue tetrazolium according to methods outlined by Flohé and Ötting (24Flohé L. Ötting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1311) Google Scholar). Following electrophoresis, SDS-containing gels were washed in 100 ml of 10 mm sodium phosphate, pH 7.8 (with or without 50 μm MnSO4), three times for 30 min each prior to SOD activity staining. Positive staining of in-gel SOD activity was performed with 4-chloro-1-naphthol. Following SDS-PAGE, gels were washed (three 20-min washes) in 10 mm MOPS, pH 7.0 (with or without 50 μm MnSO4). Staining for H2O2 production was performed by incubating the washed gels in a staining solution containing: 20 mm MOPS, pH 7.0, 28 μm riboflavin, 5 units/ml horseradish peroxidase (Sigma), 500 ng/ml 4-chloro-1-naphthol, 10 mmTEMED, and 60% ethanol. Gels were incubated in staining solution in transparent trays on a light box with gentle shaking. Staining was performed for 16–24 h. PAS staining following SDS-PAGE (28Zacharius R.M. Zell T.E. Morrison J.M. Woodlock J.J. Anal. Biochem. 1969; 30: 148-152Crossref PubMed Scopus (1678) Google Scholar) was used to examine nectarin I glycosylation. MALDI mass spectrometry was used for determining the molecular mass of the purified nectarin I protein. Protein samples of 1–2 μl containing approximately 2–4 ng of protein were loaded with 1–2 μl of freshly prepared sinapinic acid matrix onto a time-of-flight mass analyzer (Lasermat 2000 MALDI; Finnigan, Madison, WI). The collected data were analyzed using data processing software (Lasermat 2000). Bovine serum albumin was used as an internal calibration standard. To evaluate whether nectarin I might be a superoxide dismutase, we initially examined whether raw nectar contained any superoxide dismutase activity. However, the high concentrations of ascorbate present raw nectar interferes with the SOD assay (24Flohé L. Ötting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1311) Google Scholar), so nectar proteins were precipitated from raw nectar by ammonium sulfate precipitation, and the SOD assay was performed on the partially purified nectarins. Fig. 1 demonstrates that increasing amounts of partially purified nectar proteins result in decreased superoxide-dependent reduction of cytochromec, confirming that the partially purified nectar proteins do indeed contain superoxide dismutase activity. Because superoxide dismutase activity was identified with the nectar proteins, we next attempted to determine whether this superoxide dismutase activity was associated with nectarin I. We ran aliquots of ammonium sulfate-precipitated nectar proteins on native gels and demonstrated that the major nectar protein was stained for superoxide dismutase activity with nitro blue tetrazolium (data not shown). We have demonstrated previously that even in the presence of SDS the nectarin I protein migrates as an oligomer if the protein samples were not boiled prior to SDS-PAGE (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). We reasoned that if the nonboiled nectarin I protein maintains its oligomeric quaternary structure, perhaps it might also maintain its enzymatic activity. Therefore, we also examined SDS-PAGE gels for superoxide dismutase activity. Fig.2 (lane 2) shows the protein profile of ammonium sulfate precipitated nectar proteins. When the protein samples are prepared in Laemmli buffer without boiling and run on SDS-PAGE gels, the nectarin I migrates as a 165-kDa oligomer. As shown in lane 3, Western blotting using antiserum raised against nectarin I identifies the 165-kDa nectarin I oligomer. When a duplicate gel was stained for superoxide dismutase activity (lane 4), a band of enzyme activity was observed that corresponded with the 165-kDa nectarin I protein. Thus, nectarin I has superoxide dismutase activity. We next decided to purify nectarin I and evaluate superoxide dismutase activity on the purified protein. Because GLPs are known for their thermostability (29Lane B.G. Cuming A.C. Frégeau J. Carpita N.C. Hurkman W.J. Bernier F. Dratewka-Kos E. Kennedy T.D. Eur. J. Biochem. 1992; 209: 961-969Crossref PubMed Scopus (71) Google Scholar), and heat precipitation steps are extremely good first steps in the purification of many proteins (30Bryant J. Green T.R. Gurusaddaiah T. Ryan C.A. Biochemistry. 1976; 15: 3418-3424Crossref PubMed Scopus (154) Google Scholar, 31Sommert U. Traving C. Schauer R. Glycoconj. J. 1999; 16: 425-435Crossref PubMed Scopus (12) Google Scholar, 32McManaman J. Shellman V. Wright R. Repine J. Arch. Biochem. Biophys. 1996; 332: 135-141Crossref PubMed Scopus (19) Google Scholar), we explored thermostability for the purification of nectarin I from crude nectar proteins. Ammonium sulfate-precipitated nectar proteins were resuspended in 10 mm sodium phosphate buffer, pH 7.8, and dialyzed against this same buffer. Aliquots of these nectar proteins containing 17 μg of total protein were incubated at various temperatures for 5 min and evaluated for superoxide dismutase activity. As observed in Fig. 3 (panel A), the superoxide dismutase activity of nectarin I is remarkably stable over all temperatures up to 90 °C. Above 90 °C, superoxide dismutase activity rapidly declines. We also examined the kinetics of this stability. Aliquots of nectarin I were incubated at temperatures between 80 °C and 95 °C for varying periods of time and immediately placed on ice. The remaining activity of superoxide dismutase was evaluated. As shown in Fig. 3 (panel B), nectarin I shows remarkably stable superoxide dismutase activity at temperatures of 90 °C and below. Even for periods as long as 1 h at 90 °C, 85% of superoxide dismutase activity is retained. When we evaluated the protein profile of the heat-treated, ammonium sulfate precipitated nectar proteins, we were surprised to find that this single thermal denaturation step resulted in the precipitation of all nectar proteins except for nectarin I. This resulted in a two-step, near quantitative purification of nectarin I. We moved the thermal denaturation step prior to the ammonium sulfate precipitation to reduce manipulations. As can be seen in Table I, the recovery of enzyme activity was nearly quantitative. A final specific activity of 2,543 units of superoxide dismutase activity/mg of protein was found for the purified protein. This level of specific activity is similar to that observed with the E. colimanganese superoxide dismutase (20Keele Jr., B. McCord J. Fridovich I. J. Biol. Chem. 1970; 245: 6176-6181Abstract Full Text PDF PubMed Google Scholar). The purity of the thermostable nectarin I preparation was evaluated by SDS-PAGE. Fig. 4 shows the protein profile of crude nectar in lanes 1 (nonboiled) and 2 (boiled) and of the purified nectarin I preparation inlanes 3 (nonboiled) and 4 (boiled). As can be observed, in nonboiled nectar, the nectarin I oligomer migrates at 165 kDa, whereas the monomer migrates at 29 kDa (comparelanes 1 and 2). The purified nectarin I preparation also gives a single 165-kDa band on the gel when nonboiled (lane 3) and a single 29-kDa band following boiling (lane 4). Based upon these observations, we concluded that nectarin I was pure. This figure also demonstrates that the oligomeric form of nectarin I binds Coomassie Blue much less effectively than the monomeric form. Each pair of these lanes, 2 and 3, or4 and 5, contains the same amount of protein, but clearly the monomeric form gives greater interaction with the Coomassie Blue stain. The SDS-PAGE analysis of nectarin I shows a molecular mass of 29 kDa. However, the MALDI-TOF analysis of purified nectarin I showed a M+ peak of 22,533 ± 58 (n = 5). The M2+ peak was also readily detected with a mass of 45,184 ± 131 (n = 5). Larger complexes are not observed. This discrepancy in molecular masses between the SDS-PAGE and MALDI likely results from the extreme stability of the nectarin I protein during electrophoresis. If the protein is not completely unfolded and coated with SDS, then the protein would be expected to run slower than expected, producing an artificially high molecular mass on the SDS-PAGE. The molecular mass of the mature nectarin I protein predicted from the amino acid sequence is 21,062 Da (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar). The difference between the predicted molecular mass and that found by mass spectrometry, 1,471 Da, is unaccounted for. However, it is known that GLPs are glycosylated (33Jaikaran A.S.I. Kennedy T.D. Dratewka-Kos E. Lane B.G. J. Biol. Chem. 1990; 265: 12503-12512Abstract Full Text PDF PubMed Google Scholar). All GLPs, including nectarin I, contain a conserved site ofN-glycosylation (2Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1999; 41: 207-216Crossref PubMed Scopus (83) Google Scholar, 10Domon J.-M. Dumas B. Lainé E. Meyer Y. Alain D. David H. Plant Physiol. 1995; 108: 141-148Crossref PubMed Scopus (67) Google Scholar, 11Carter C. Graham R. Thornburg R.W. Plant Mol. Biol. 1998; 38: 929-943Crossref PubMed Scopus (60) Google Scholar, 16Neutelings G. Domon J. Membre N. Bernier F. Meyer Y. David A. David H. Plant Mol. Biol. 1998; 38: 1179-1190Crossref PubMed Scopus (54) Google Scholar, 17Vallelain-Bindschedler L. Mösinger E. Métraux J.-P. Schweizer P. Plant Mol. Biol. 1998; 37: 297-308Crossref PubMed Scopus (81) Google Scholar, 19Ohmiya A. Tanaka Y. Kadowaki K. Hayashi T. Plant Cell Physiol. 1998; 39: 492-499Crossref PubMed Scopus (58) Google Scholar, 33Jaikaran A.S.I. Kennedy T.D. Dratewka-Kos E. Lane B.G. J. Biol. Chem. 1990; 265: 12503-12512Abstract Full Text PDF PubMed Google Scholar). PAS staining (28Zacharius R.M. Zell T.E. Morrison J.M. Woodlock J.J. Anal. Biochem. 1969; 30: 148-152Crossref PubMed Scopus (1678) Google Scholar) demonstrated the presence of carbohydrate on the purified nectarin I protein (Fig. 4, lanes 6 and 7). Jaikaran et al. (33Jaikaran A.S.I. Kennedy T.D. Dratewka-Kos E. Lane B.G. J. Biol. Chem. 1990; 265: 12503-12512Abstract Full Text PDF PubMed Google Scholar) have reported the structure of theN-linked glycan from wheat germin. That structure is a biantennary nonasaccharide with the composition (GlcNAc)4:Man3:Xyl:Fuc. This nonasaccharide has a molecular mass of 1,576 Da, which corresponds well with the mass differences observed between the MALDI-TOF analysis and the cDNA-predicted molecular mass (1,471 Da). Therefore, we expect that the nectarin I glycan is highly similar to the N-linked glycan present on wheat germin. To determine whether the purified nectarin I had superoxide dismutase activity, we next evaluated the ability of the purified nectarin I to remove superoxide generated by xanthine-xanthine oxidase. As can be seen in Fig. 5, the purified protein was indeed able to dismute superoxide in a dose-dependent manner. Therefore, we conclude that the superoxide dismutase activity associated with tobacco nectar is due to the presence of nectarin I. Based upon the type of metals that they contain, there are three known families of superoxide dismutases: FeSOD, Cu/ZnSOD, and MnSOD. To determine the type of superoxide dismutase family to which nectarin I belongs, we analyzed the purified nectarin I protein for metal ions. This analysis demonstrated the presence of 0.5 mol of manganese/mol of nectarin I monomer. Iron and copper were present in trace amounts at or near the limits of detection. To confirm that nectarin I was a manganese superoxide dismutase, hydrogen peroxide inhibition studies of enzyme activity were performed. Manganese superoxide dismutases are stable in the presence of 5 mm H2O2, whereas copper/zinc and iron superoxide dismutases lose activity following this treatment (34Koster J.F. Slee R.G. Van Berkel T.J.C. Bannister W.H. Bannister J.V. Biological and Clinical Aspects of Superoxide and Superoxide Dismutase. Elsevier, New York1980Google Scholar,35Geller B.L. Winge D.R. Anal. Biochem. 1983; 128: 86-92Crossref PubMed Scopus (95) Google Scholar). Because nectarin I retains superoxide dismutase activity following SDS-PAGE (see Fig. 2, lane 3), we examined this inhibition following gel electrophoresis. Lanes 1and 3 of Fig. 6 contain a mixture of commercially available superoxide dismutases, including the manganese superoxide dismutase from E. coli (20Keele Jr., B. McCord J. Fridovich I. J. Biol. Chem. 1970; 245: 6176-6181Abstract Full Text PDF PubMed Google Scholar), the iron superoxide dismutase from E. coli (21Yost F.J. Fridovich I. J. Biol. Chem. 1973; 248: 4905-4908Abstract Full Text PDF PubMed Google Scholar), and the copper/zinc superoxide dismutase from bovine erythrocytes (22Steinman H. Naik V. Abernethy J. Hill R. J. Biol. Chem. 1974; 249: 7326-7338Abstract Full Text PDF PubMed Google Scholar). Lanes 2 and 4 contain purified nectarin I. The nonboiled proteins were all separated on SDS-PAGE gels and stained for superoxide dismutase activity with nitroblue tetrazolium (24Flohé L. Ötting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1311) Google Scholar). As can be observed in lanes 1 and 2, each of the nonboiled proteins retains superoxide dismutase activity following SDS-PAGE. Treatment of these proteins for 1 h with 5 mm H2O2, however, results in the loss of activity of the iron and copper/zinc superoxide dismutases. In contrast, both the manganese superoxide dismutase from E. coli and the nectarin I superoxide dismutase remain active following this treatment. Similarly, NaCN is capable of inactivating Cu/Zn superoxide dismutases (34Koster J.F. Slee R.G. Van Berkel T.J.C. Bannister W.H. Bannister J.V. Biological and Clinical Aspects of Superoxide and Superoxide Dismutase. Elsevier, New York1980Google Scholar, 35Geller B.L. Winge D.R. Anal. Biochem. 1983; 128: 86-92Crossref PubMed Scopus (95) Google Scholar). Incubation with NaCN did not inhibit the enzymatic activity of nectarin I (data not shown). Although nonboiled nectarin I retains its quaternary structure and its superoxide dismutase activity during SDS-PAGE, it decomposes to its monomeric form following boiling. This monomeric form does not retain the superoxide dismutase activity after boiling (comparelanes 1 and 2 of Fig.7). We therefore decided to test whether we could reactivate the superoxide dismutase activity following metal ion replacement. As shown in Fig. 7, the addition of 50 mmFeSO4, CuSO4, ZnCl2, or CuSO4/ZnCl2 to the gel wash solutions failed to reactivate the superoxide dismutase activity (lanes 3, 4, 5, or 6). In contrast, addition of 50 mm MnSO4 produced active enzyme (lane 7). Thus, only manganese was able to reconstitute enzyme activity, confirming that the nectarin I is a manganese superoxide dismutase. The studies illustrated in Fig. 7 do not provide information about the quaternary structure of the active form of nectarin I following metal ion replacement. To examine this in more detail, we first inactivated the superoxide dismutase activity by boiling. After separating the monomeric form on an SDS-PAGE gel, we renatured the enzyme as inlane 7 of Fig. 7. The renaturation was verified by staining the gel for superoxide dismutase activity (data not shown). Subsequent" @default.
• W2023160617 created "2016-06-24" @default.
• W2023160617 creator A5027506691 @default.
• W2023160617 creator A5083522225 @default.
• W2023160617 date "2000-11-01" @default.
• W2023160617 modified "2023-10-11" @default.
• W2023160617 title "Tobacco Nectarin I" @default.
• W2023160617 cites W1490647236 @default.
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