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First published online October 21, 2005; 10.1104/pp.105.065227 Plant Physiology 139:1389-1400 (2005) © 2005 American Society of Plant Biologists Nectarin IV, a Potent Endoglucanase Inhibitor Secreted into the Nectar of Ornamental Tobacco Plants. Isolation, Cloning, and Characterization1,2Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 (S.M.S.N., C.C., G.R., A.G., R.W.T.); and Department of Biochemistry, Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30601 (A.H., W.S.Y.)
We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii x Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5' and 3' rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a Ki of 0.35 nM. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.
Many higher plants have evolved unique strategies for successful fertilization and propagation that rely on animal-mediated pollen transfer. A metabolically rich nectar reward is one of the most important factors in this strategy. The attraction of these pollinators results in a more efficient transfer of pollen from flower to flower, increasing genetic out-crossing and fecundity. The nectar produced by most plants is a rich combination of substances, primarily Suc, Glc, and Fru. However, other carbohydrates occur in some nectars (Baker and Baker, 1981  ). All 20 normal amino acids have been identified in various nectars (Baker and Baker, 1973). Other substances include organic acids (Baker and Baker, 1975), terpenes (Ecroyd et al., 1995), alkaloids (Deinzer et al., 1977), flavonoids (Rodriguez-Arce and Diaz, 1992), glycosides (Roshchina and Roshchina, 1993), vitamins (Griebel and Hess, 1940), phenolics (Ferreres et al., 1996; Cabras et al., 1999), oils (Vogel, 1969), and five proteins, termed nectarins (Carter et al., 1999; Thornburg et al., 2003).
We have previously characterized four of the five nectar proteins (Carter et al., 1999 In this study, we have isolated and characterized the cDNA encoding NEC4 and have demonstrated that the NEC4 protein functions as a xyloglucan-specific fungal endoglucanase-inhibitor protein (XEGIP). This confirms that this nectar protein is also a defense-related protein. Because NEC4 was the last of the nectar proteins to be isolated and characterized, these studies have permitted us to complete the characterization of the entire tobacco nectar proteome and have allowed us to demonstrate that the primary function of each of the nectar proteins is in plant defense or stress responses.
Identification of NEC4
Because automated Edman degradation of NEC4 isolated from SDS-PAGE gels does not identify any N-terminal sequence, we had previously concluded that the NEC4 N terminus is apparently blocked (Carter and Thornburg, 2004a
Cloning of the NEC4 cDNA To isolate the NEC4 cDNA, we utilized a strategy that was based upon the common identities of the tomato and potato XEGIP homologs. ClustalW pileup analysis of the gene sequences encoding the proteins AAN87262 and AAP84703 (accession nos. AY155579 and AY321357) permitted us to identify conserved nucleotide sequences and to design a pair of conserved oligonucleotide primers for PCR amplification. These oligonucleotides were used to generate a PCR product from an ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae var LxS8) nectary cDNA library. The PCR product was cloned into pGEM-T. This partial cDNA clone (pRT537) was sequenced to generate additional oligonucleotides used together with oligonucleotides designed from library vector sequences to amplify the 5' and 3' ends of the cDNA from a stage 6 nectary cDNA library. After the first round and a nested round of PCR, the 3' RACE identified two independent nearly full-length clones that contained the 3' ends of the NEC4 clone (pRT540 and 541). These 3' ends were >99% identical. They differed by three single nucleotide changes (C845, G994, and T1094 of pRT540 were T845, A994, and C1094 in pRT541) and a single 32-bp deletion in the 3' untranslated region of pRT540 relative to pRT541 at positions 1,298 to 1,330. The single nucleotide changes were all located in the coding region and each of them results in amino acid changes: Asp-289, Val-339, and Val-372 in pRT540 were Val-289, Thr-339, and Ala-372 in pRT541. The polyA addition sites also differed between these two clones; pRT540 was 48 nucleotides longer than pRT541. These cDNA differences may represent recently diverged, closely related genes that are similarly expressed; however, because the plants used in this study were derived from an interspecific cross between two closely related tobacco species, the two sequences may represent the NEC4 sequences from these two species. The 5' RACE identified a number of short, identical clones, one of which was selected as pRT544.
Comparisons of the isolated NEC4 cDNA sequences with the 5' and 3' RACE sequences permitted us to generate a full-length NEC4 cDNA. This sequence was deposited in GenBank (accession no. AY898762). This 1,618-nucleotide composite cDNA contains no significant hairpins or repeat elements. The 5' and 3' untranslated regions are 36 and 237 nucleotides, respectively. The cDNA encodes a 437-amino acid protein with high identity to the tomato and potato XEGIPs. The NEC4 protein shares 88.8% identity with the potato clone (AY321357) and 88.3% identity with the tomato clone (AY155579). The potato and tomato clones share 96.6% identity. To further confirm that the NEC4 cDNA encoded the NEC4 protein, NEC4 was isolated from floral nectar and subjected to tryptic peptide mass fingerprinting. This analysis (Table II) confirmed the presence of additional peptides that were encoded by the NEC4 cDNA. Together with the sequences of the three peptides that were sequenced from the NEC4 protein, this confirms that 28.7% of the NEC4 coding region corresponds to amino acid sequences in the mature NEC4 protein.
NEC4 N Terminus Examination of the N-terminal sequence of the NEC4 protein (McGeoch, 1985 ; Nakai and Kanehisa, 1991) revealed that the NEC4 N terminus likely contains a 22-amino acid signal sequence (Fig. 1). This is consistent with the extracellular localization of the NEC4 protein. This sequence is also similar to the signal sequences of each of the other tobacco nectarins. After cleavage of the N-terminal signal sequence at Ala-22, the resulting N-terminal amino acid is Gln (Gln-23), which is consistent with the presence of a pyroglutamyl-blocked N terminus that is refractory to Edman degradation. Together with the identification of almost 30% of the mature NEC4 coding region from the NEC4 protein (Fig. 1; Tables I and II), the finding that the NEC4 clone should also have a blocked N terminus convinced us that the NEC4 cDNA did indeed encode the NEC4 protein from the nectar of ornamental tobacco.
NEC4 Model Using the amino acid sequence of the NEC4 protein, we searched the Protein Data Bank (PDB) to identify similar proteins. One protein, the wheat (Triticum aestivum) xylanase inhibitor (PDB no. 1T6E), showed 29.1% identity and 53.4% similarity to the protein sequence predicted from the NEC4 cDNA. Using the coordinates of the wheat xylanase inhibitor TAXI-1, a model was built by threading the NEC4 amino acid sequence through those coordinates. The model was optimized using the backbone atoms only, and, after two rounds of structural alignment, the optimization procedure produced a usable model. This model is based on 399 amino acids of the NEC4 sequence from Lys-31 to Cys-428, and contained 1,292 protein backbone atoms that showed a root mean square (RMS) deviation of 0.57 Å from the wheat xylanase inhibitor. The models are shown in Figure 2. The most disordered regions in the tobacco model relative to the wheat inhibitors corresponded to six loops caused by insertions of between three to 10 amino acids in the tobacco sequence (shown as red residues in Fig. 2, A and B). These loops are not present in the wheat xylanase inhibitor. If we excluded these loops from the NEC4 model, the RMS deviation of the remaining 1,228 backbone atoms dropped to 0.40 Å, indicating that the remainder of the molecular conformation is well conserved between the two proteins. Subsequently, a second model was built using the wheat xylanase inhibitor complexed with the Aspergillus niger xylanase I (1T6G). In this second model, the optimized NEC4 protein model was overlaid onto the structure of the wheat xylanase inhibitor (Fig. 2C). This model also contained 399 amino acids of the optimized NEC4 protein sequence and the 1,300 NEC4 backbone atoms that showed a RMS deviation of 0.61 Å with the wheat inhibitor.
The yellow region of Figure 2A shows the amino acids that are homologous to the TAXI-1 xylanase-binding site. The loops (red) and sites of glycosylation (green) are also shown on the model (Fig. 2, A and B). Interestingly, all of the loops and carbohydrates are on the opposite face of the NEC4 molecule from the region that is homologous to the TAXI-1 xylanase-binding site. Thus, we expect that these loops and glycosylation sites would not interfere with interactions with the glycosylhydrolase ligand, assuming its interaction with NEC4 is similar to the TAXI-1 xylanase interaction. Alternatively, the possibility exists that these loops could be alternative binding sites for other glycosylhydrolases.
The TAXI-1 molecule contains a knottin-like domain (Sansen et al., 2004
Further, like TAXI-1, the NEC4 molecule also has overall structural homology with aspartic proteases (Sansen et al., 2004
NEC4 Glycosylation
NEC4 Phylogenetic Relationships
Based upon this analysis, we can derive several features about the biology of this gene family. First, the 13 rice (Oryza sativa) homologs identify two islands of tandemly repeated genes within the rice genome. One is present on the bacterial artificial chromosome clone 006J12 (four sequences). The other is present on the bacterial artificial chromosome clone 504E02 (eight sequences). Similarly, five of the six Arabidopsis NEC4 homologs are clustered in a pair of tandem arrays on chromosomes 1 and 5.
Second, the gene sequences from the Fabaceae were previously identified as seed storage proteins in lupins (Lupinus sp.), chickpea (Cicer arietinum), and soybeans (Glycine max; York et al., 2004 Finally, NEC4 shows significant homology with the wheat xylanase inhibitors TAXI-1 through TAXI-4 (E-scores < e–37). The identification of hemicellulase inhibitor activity in gene products in widely separated clades of this phylogenetic tree (NEC4 and TAXI-1) suggests that the ability to inhibit fungal glycan hydrolases may be a common feature of all members of this gene family and that inhibition of fungal hemicellulases may be a much more important feature of plant defense than has been previously recognized. Further, the identification of NEC4-related sequences from six different plant families (Apiaceae, Brassicaceae, Fabaceae, Poaceae, Rosaceae, and Solanaceae) confirms that these genes are widely dispersed throughout the angiosperms.
Because NEC4 has such high identity with a known xyloglucan-specific endoglucanase inhibitor, it was important to determine whether the NEC4 protein also can act as a glucanse inhibitor. Therefore, we purified the NEC4 to homogeneity so that we could test it for XEGIP and other inhibitory activities. The simplicity of the nectar proteome (only five proteins accumulate in nectar; Carter et al., 1999
NEC4 Is a XEGIP
To test for inhibition, aliquots of purified NEC4 were incubated with a xyloglucan-specific endoglucanase (XEG) from Aspergillus aculeatus (Pauly et al., 1999
Expression of NEC4 To evaluate the temporal and spatial expression patterns of the NEC4 gene, we examined the levels of NEC4 mRNA by reverse transcription (RT)-PCR. mRNA was isolated from a number of floral and nonfloral plant organs, reverse transcribed, and used as a template for PCR. As a control, we used a pair of glyceraldehyde-3-P dehydrogenase (GAPDH) oligonucleotides. The GAPDH mRNA showed an equal level of expression in each of the organs examined (Fig. 6, all lanes). In contrast, the NEC4 oligonucleotides showed expression only in the nectary tissues. No other floral organ, nor roots, stems, or leaves, showed expression of the NEC4 mRNA. Temporally, NEC4 showed no expression at the earliest stages of nectary development (Fig. 6, lanes 1 and 2). By stage 9 (presecretory, ripening nectaries), a low level of expression was observed (Fig. 6, lane 3). This level of expression increased with further development until a moderate level was observed by stage 12 (mature nectaries at anthesis; Fig. 6, lane 4). Surprisingly, the level of NEC4 showed an increased expression after pollination. Figure 6, lane 5, shows an extremely high level of expression 48 h after fertilization.
We have isolated and characterized a 64-kD protein, NEC4, from the nectar of ornamental tobacco plants. This protein is unique among the known nectar proteins. It has a blocked N terminus. To generate protein sequence in preparation for cloning, we used MS sequencing of NEC4 tryptic peptides. Three peptides were identified that closely matched a tomato protein (AAN87262) or its potato homolog (AAP84703). Alignment of the cDNAs encoding these two proteins permitted the identification of conserved regions that were used to generate oligonucleotide primers. PCR was performed on an ornamental tobacco nectary cDNA library to isolate a partial cDNA clone. 5' and 3' RACE were used to generate a full-length clone, which was deposited in GenBank (accession no. AY898762).
The N-terminal sequence of the NEC4 protein likely contains a 22-amino acid signal sequence that, when cleaved, results in an N-terminal Gln residue (Gln-23). It is of interest to note that N-terminal Gln residues are frequently converted into pyroglutamate by specific Gln cyclase enzymes (Oberg et al., 1998 Based upon similarities with the wheat xylanase inhibitor (TAXI-1), we were able to model the structure of the NEC4 protein. The models produced in this study permitted us to identify a number of structural features that may be related to NEC4 function. We found that the NEC4 protein contains six small loops caused by the insertion of between three to 10 amino acids that are not found in the TAXI-1 molecule. We also identified the sites of glycosylation on the model. Each of these loops and glycosylation sites are all located well away from the region that is homologous with the TAXI-1 xylanase-binding site and would not be expected to interfere with glycohydrolase binding. This model also permitted us to identify a modified knottin domain that was also located well away from the glycohydrolase-binding site.
Knottin domains have been proposed to be sites mediating protein-protein interactions (Smith et al., 1998
The identification of closely related NEC4 homologs demonstrated that NEC4 belongs to a moderately sized multigene family. Rice contains 13 NEC4 homologs and Arabidopsis contains six homologs. Based upon the genome sequences of rice and Arabidopsis, we observed that the NEC4 family members are located on islands of tandemly repeated genes, as is found with other plant defense genes (Wei et al., 2002
As was previously observed, three main classes of genes were identified in this analysis: XEGIP-related proteins, TAXI-related proteins, and seed storage proteins (York et al., 2004
NEC4 was purified from ornamental tobacco nectar using a one-step HPLC method. The NEC4 protein was subsequently shown to tightly bind a GH12 family XEG. The glycosylhydrolase family GH12 (Coutinho and Henrissat, 1999
Endoglucanases represent a large family of fungal proteins that degrade plant cell walls (Collins et al., 2005
The finding that NEC4 could inhibit GH12 endoglucanases, but was unable to inhibit GH11 xylanases, is intriguing. Family GH11 xylanases have overall folds that are similar to GH12 endoglucanases. In contrast, family GH10 xylanases have a different folding pattern than either the GH11 or GH12 hydrolases and are inhibited by a different wheat protein called XIP-I (Payan et al., 2004
Because NEC4 is a potent inhibitor of fungal endoglucanases, the pattern of NEC4 expression suggests a unique feature about the biochemistry of this protein. The protein is increasingly expressed as the nectary develops. However, it is most highly expressed after fertilization. Thus, it appears that inhibition of fungal hemicellulases may be more important after fertilization than it is at anthesis. Examination of other plant organs indicates that the NEC4 protein is not expressed in any other plant organ tested. Thus, the NEC4 promoter may be very valuable for future studies to express novel genes during the late stages of nectary development.
The nectar of ornamental tobacco contains a limited array of five proteins. NEC4 was the last of the tobacco nectar proteins to be identified and characterized. Thus, the characterization of NEC4 as an inhibitor of XEG completes the analysis of the Nicotiana nectar proteome. The analysis of these novel proteins has permitted us to identify several unique biochemical functions of nectar (Table III). First, we have identified that the nectar of Nicotiana plants contains very high levels of hydrogen peroxide (Carter and Thornburg, 2000
We have previously proposed that a heretofore unrecognized function of the nectary is the protection of the gynoecium from pathogen invasion (Thornburg et al., 2003 ). The finding of a complex defense system in nectar suggests a novel function that has been a previously unrecognized function of nectar, that the secreted nectar not only functions to attract insect pollinators, but it also functions to protect the gynoecium from microbial invasion of the metabolite-rich nectar (Thornburg et al., 2003). This finding is similar to the finding that other defense-related proteins also accumulate in other floral organs and are also thought to protect flowers and presumably the gynoecium from infection (Lotan et al., 1989; Lay et al., 2003). Our identification of NEC4 as a potent inhibitor of fungal endoglucanases provides strong support for this hypothesis. Further, the expression pattern of NEC4 mRNA implies that the nectary continues this protective function even after pollination has already occurred.
Plants
The line of ornamental tobacco plants used in this study was derived from an interspecific cross between Nicotiana langsdorffii and Nicotiana sanderae (Kornaga et al., 1997
Bands from an SDS-PAGE gel containing NEC4 were excised and processed for MS analysis as described previously (Wang et al., 2000
MALDI-TOF
Database Searches
Cloning methods were conducted either according to the manufacturer's directions or by accepted methods (Sambrook et al., 1989
Nectary cDNA Library
cDNA Library Screening
5' and 3' RACE For amplification of the remaining 3' end of the cDNA, plasmid DNA isolated from the stage 6 nectary cDNA library was amplified using Nec4F2 (5'-GGGTTGATTGTGACCAAGGT-3') and M13-R (5'-CAGGAAACAGCTATGACC-3') oligonucleotides. The amplified product was diluted 100-fold and used as template for nested amplification using Nec4F3 (5'-GGGAGAACCCTCTTCTGAAT-3') and oligo(dT)25V (5'-TTTTTTTTTTTTTTTTTTTTTTTTTV-3'). PCR products were processed for cloning as for 5' RACE.
Plant mRNA was isolated from various floral and nonfloral organs using the SV total RNA isolation kit (no. Z3500) according to the manufacturer's directions (Promega). This RNA was used for first-strand cDNA synthesis using the StrataScript first-strand cDNA synthesis kit (no. 200420; Stratagene). The cDNA was aliquoted and stored at –70°C until use. RT-PCR was performed using the first-strand cDNA and oligonucleotides specific for the coding region of NEC4, nec4bF (5'-GGCTGGTCTTGGTAGGACAAGAATAT-3') and nec4bR (5'-GATTGTCAATGGACAACAAAGTTGTG-3'). The LxS8 GAPDH mRNA (expressed sequence tag clone 101G06) isolated from an expressed sequence tag study of nectary-expressed genes was used as an internal control. GAPDH oligonucleotides GAP2F (5'-TTGAAGGGTGGTGCCAAGAAA-3') and GAP2R (5'-CATTTATGACCTTTGCCAAGGGAG-3') were specific for the GAPDH coding regions. PCR products were run on a 1.5% agarose gel and visualized using ethidium bromide.
Ten milliliters of nectar were collected over a 1-h time frame and were maintained on ice during this process. Following collection, the nectar was passed through a 0.2-µm filter and then subjected to concentration through Centricon YM-10 centrifugal columns at 4,000g until the retentate volume was reduced to about 1/20. The concentrated nectar was then diluted 10-fold with 10 mM HEPES buffer, pH 7.3, and recentrifuged to concentrate the volume to less than 100 µL. The retentate was again diluted 20-fold and used as an HPLC sample. Aliquots (750 µL) of this sample were injected into HPLC in buffer A (10 mM HEPES, pH 7.3) and analyzed through a cation exchange column CM300-silver (250 mm x 4.6 mm i.d.). The elution profile included the first 20 min with buffer A. This was followed by a linear gradient from 100% buffer A to 100% buffer B (10 mM HEPES, pH 7.3, 400 mM NaCl) followed by another 30 min with 100% buffer B. The flow rate was kept constant at 0.5 mL/min. The elution profile was monitored at 280 nm. Fractions pertaining to the various peaks were separately pooled and concentrated by Centricon YM-10 centrifugal columns. Protein concentrations were estimated by Bradford (1976)
Protein models were developed using SWISS MODEL (Peitsch, 1995
The purified NEC4 protein was diluted with buffer C (50 mM sodium acetate, pH 4.7) to yield a 50.0 nM solution, as determined using an extinction coefficient (
The resulting data were fit to a theoretical curve in which each value of the dependent variable vi (the reaction velocity in the presence of inhibitor) is a function of the independent variable [I]t (the total inhibitor concentration), and the parameters Ki,app (the apparent inhibition constant) and [E]t (the total enzyme concentration), as illustrated in figure III-29 of Segel (1975)
A theoretical data curve was constructed by calculating theoretical (not necessarily integer) values for n based on the independent variable [I]t and the adjustable parameters Ki,app and [E]t. That is, n was determined for each experimental [I]t, allowing the corresponding theoretical value of vi,theor to be calculated. The calculated values of vi,theor were compared to the corresponding experimental value of vi, and trial values of Ki,app and [E]t were adjusted until the RMS value of the residuals was minimized.
Several xylanases were tested for inhibition by adding xylanase to a solution containing substrate (xylan) and a stoichiometric excess of the NEC4 protein and colorimetrically measuring the enzyme-catalyzed increase in reducing residues. Enzymes that were assayed included Trichoderma viride xylanase M1 (Megazyme International Ireland), which is a family GH11 xylanase, three family GH11 xylanases from Magnaporthe grisea that were heterologously expressed in Pichia pastoris, and one family GH10 xylanase purified from M. grisea strain CP987 (Wu et al., 1997 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY898762.
We thank Dr. Natasha Raikhel and Songqin Pan for technical assistance and the generous use of instrumentation housed in the W.M. Keck Proteomics Laboratory, Center for Plant Cell Biology, University of California, Riverside, and Novozymes A/S, who generously provided the xyloglucan-specific endoglucanase used in this research. Received May 9, 2005; returned for revision August 15, 2005; accepted September 12, 2005.
1 This work was supported by the National Science Foundation (grant no. IBN–0235645); the Carver Trust, the Hatch Act, and State of Iowa Funds; a visiting fellowship from the Higher Education Commission of Pakistan (to S.M.S.N.); and in part by funds from the U.S. Department of Energy (DOE; grant no. DE–FG02–96ER20220) and from the DOE-funded Center for Plant and Microbial Complex Carbohydrates (grant no. DE–FG02–93ER20097). 
2 This paper is dedicated with grateful appreciation to Professor C.A. "Bud" Ryan, Institute of Biological Chemistry, Washington State University, on the occasion of his 74th birthday, September 29, 2005.
3 Present address: Department of Biochemistry, University of Arid Agriculture, Rawalpindi 46300, Pakistan.
4 Present address: Department of Biology, University of Minnesota, Duluth, MN 55812.
5 Present address: Institute of Genetic Engineering and Biotechnology, Menofiya University, Sadat City, Egypt. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert W. Thornburg (thorn{at}iastate.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.065227. * Corresponding author; e-mail thorn{at}iastate.edu; fax 515–294–0453.
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ABSTRACT
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backbone (in blue). D, Knottin domain of TAXI-1 in blue overlaid with the pseudoknottin domain of NEC4 (in yellow). The three disulfide linkages are color coded (red, orange, and purple). E, Amino acid sequences of the knottin domain of TAXI-1 and the corresponding pseudoknottin domain of NEC4. Cys residues are highlighted and disulfide linkages are color coded to match those shown in D.
clone 32 (white lupin), 
-galactosidase, 116 kD; phosphorylase b, 97 kD; bovine serum albumin, 66 kD; ovalbumen, 45 kD; carbonic anhydrase, 31 kD; soybean trypsin inhibitor, 21.5 kD; and lysozyme, 14.4 kD); lane 2, raw nectar; lane 3, proteins from pool 1; lane 4, proteins from pool 2.
where n is an integer. The distance (0.45 nm) between successive intercepts of these lines with the [I]t axis is an estimation of Ki,app. The competitive inhibition constant Ki is calculated as follows: 
) of 82,123 M–1cm–1 (Qin et al., 2003
A diagonal line passing through the points (0, v0) and ([I]t, vi) will intercept the [I]t axis at a value 
For graphical convenience, the original formulation of this approach limited n to integer values, allowing Ki,app to be estimated as the distance between successive intercepts. Examination of figure III-29 of Segel (1975)
