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1 TOYOBO Co. Ltd. Bio 21 Project, 10-24 Toyo-Cho, Tsuruga, Fukui 914-0047, Japan
2 Centre for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
3 Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan
4 Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA
5 ERATO Environmental Response Project, Japan Science and Technology Corporation, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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The Maf family proteins appear to be typical members of a large group of regulatory factors characterized by a basic region and leucine zipper (bZip) structure (Motohashi et al. 2002). The founding member of this family, v-Maf, is an oncogene, which was discovered as the transforming component of the avian musculoaponeurotic fibrosarcoma virus, AS42 (Nishizawa et al. 1989). Subsequently, it was found that the cellular homologue, from which v-Maf was originally transduced (c-Maf), was but one member of a multigene family of related transcription factors. To date, this family consists of four large Maf family members, c-Maf, MafB, NRL, and L-Maf/A-Maf, and three small Maf proteins, MafF, MafG, and MafK (Kataoka et al. 1994b, 1995; Swaroop et al. 1992; Ogino & Yasuda 1998; Fujiwara et al. 1993). The proteins interacting with the small Maf family members have been expanding to include new members in Cap’n’collar (CNC) and Bach families: p45 NF-E2, Nrf1/LCR-F1, Nrf2/ECH, Nrf3, Bach1, and Bach2 (Andrews et al. 1993; Chan et al. 1993; Moi et al. 1994; Itoh et al. 1995; Kobayashi et al. 1999; Oyake et al. 1996). The superficially arbitrary division of the Maf family into small and large members is likely of functional consequence, since all of the large Mafs appear to contain a recognizable transactivation domain, while the small Mafs encode slightly more than the DNA binding and dimerization motifs.
The bZip domain of the Maf factors are characterized by the presence of extended homology region (EHR), which is located in the N-terminal side of the basic region (Swaroop et al. 1992; ancillary DNA binding region, Kerppola & Curran 1994). DNA-binding specificity of the Maf family factors was determined by PCR-gel mobility shift assay (GMSA) amplification and purification method (Kerppola & Curran 1994; Kataoka et al. 1994a). Conclusion of these studies are that Maf factors recognize relatively long palindromic DNA sequences, TGCTGAG/CTCAGCA and TGCTGAGC/CGTCAGCA, which are now known as Maf recognition elements (MAREs). MAREs contain either TPA-responsive element (TRE; TGAG/CTCA) or cAMP-responsive element (CRE; TGAGC/CGTCA) as a core sequence, and extended elements on both sides of the core sequence (flanking region; 5'-TGC-core-GCA-3'). The recognition of the flanking region in MARE by EHR distinguishes the Maf family proteins from members of the AP-1 or CREB family of the bZip transcription factor superfamily. Kerppola & Curran (1994) showed evidence that the consensus sequence of large Maf binding is TGC(N)6-7GCA. Since the flanking region of MARE is consistently required, the study strongly suggests an important contribution of the flanking region to the Maf-specific DNA-binding. Indeed, we showed that Maf EHR is important for the flanking region recognition (Kusunoki et al. 2002). It has also been reported through amino acid replacement/mutation analysis that a unique amino acid in the basic region is involved in the flanking region recognition by Maf proteins (Dlakic et al. 2001).
Currently, GMSA is a standard method to examine the interaction between transcription factors and DNA motifs and to obtain an equilibrium constant. However, GMSA is a low-throughput method for quantification of the interaction, which usually requires labourious sample preparation steps. Recently, electrodes (Boon et al. 2002) and surface plasmon resonance (SPR, Jost et al. 1991; Suzuki et al. 1998) techniques have been developed, and these techniques are exploited to analyse the interaction between surface immobilized molecules and those in solution. Especially, SPR has advantages that it does not require any labelled reagents and can be applied for the wide surface area (Jordan & Corn 1997). The SPR technique is especially useful for a semiquantitative analysis, as it detects a dynamic real-time interaction profile.
Another recent progress has been made in the field of chip technology, which has been applied for the study of various interactions among proteins and nucleic acid fragments as microarrays (Schena et al. 1995; MacBeath & Schreiber 2000; Zhu et al. 2001; Bulyk et al. 2001; Newman & Keating 2003). Indeed, Nelson et al. (1999) developed a prototype of imaging technique for the detection of the biomolecular interaction by combining the SPR and chip technology. This SPR imaging technique seems to enable us to analyse multiple protein-DNA interactions simultaneously and comprehensively. In this respect, SPR is more advantageous than the methods exploiting electrodes upon combination with the chip technology for a comprehensive analysis, since it would be very labourious to construct an array of tiny electrodes on a chip.
Although a multistep array fabrication procedure has been developed for the SPR-chip imaging to detect the protein-DNA interactions (Brockman et al. 1999), application of this technology has been hampered due to technical difficulties. In particular, double-stranded DNAs could not be directly immobilized on the chip surface, as organic solvent used in the original procedure easily denatures delicate biomolecules. In the previous procedure (Brockman et al. 1999), single-stranded oligonucleotides were first attached on to the gold surface followed by the hybridization with the complementary DNAs to generate double-stranded DNAs on the chip. However, in order to perform a comprehensive affinity quantification of transcription factors to various suboptimal sequences, it is required to fabricate a double-stranded DNA array composed of multiple sequences that are very similar to one another. Immobilization of preannealed double-stranded DNAs is highly preferable for preventing mismatched hybridization and for assuring complete pairing between complementary DNAs.
To develop an efficient and reliable method to detect specific protein-DNA interactions exploiting the SPR technology, we have designed in this study a modified multistep procedure for generation of DNA array on the gold surface, which does not require steps exposing DNA to noxious organic solvents. We also found a better heterobifunctional crosslinker that reduces nonspecific adsorption of the protein to the chip surface in the immobilization process. By utilizing the SPR imaging technique with the newly developed double-stranded DNA array, we then examined binding affinity of MafG, one of the small Maf family members, to several MARE-related DNA sequences. The relative affinities between MafG, various MARE-related sequences showed a very good correlation to those obtained from GMSA. Thus, the new surface immobilization procedure has enabled various delicate biomolecules, including double-stranded DNAs, to be attached stably on to the gold chip in their native form. This procedure provides a solid basis for the study of SPR-based protein-DNA interactions.
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Results |
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A seven-step fabrication procedure has been used for the immobilization of biomolecules on the gold surface (Brockman et al. 1999), which was based on self-assembled monolayers (SAMs) of alkanethiol (Troughton et al. 1988; Chidsey & Loiacono 1990) and photolithography technique (Tarlov et al. 1993; Huang et al. 1994). In the procedure, the hydrophobic protecting group, 9-Fluorenylmethoxycarbonyl (Fmoc), was used for the background protection, and it was deprotected by weak base in organic solvent after single-stranded DNA was immobilized on the surface. In the final step of this procedure, an N-hydroxysuccinimido ester poly(ethylene glycol) (NHS-PEG) was reacted to an amino group on the surface. In these processes, the immobilized single-stranded DNAs were exposed to organic solvents and NHS-PEG.
In order to avoid exposure of test biomolecules to noxious effect, we established a modified procedure to fabricate double-stranded DNA array on the gold surface using thiol-terminated methoxypoly(ethylene glycol), PEG-thiol. This procedure consists of 5 steps described in Fig. 1. Step 1 is the PEG-thiol immobilization on the whole surface area of a gold slide; Step 2 is the photo-patterning by UV irradiation shielded with a bored chromium quartz mask; Step 3 is the introduction of amine terminated alkanethiol on the irradiated spots; Step 4 is the creation of maleimido surface on the spots by reacting with a heterobifunctional crosslinker, which contains a NHS ester and a maleimido group; Step 5 is the 5'-thiol-terminated DNA immobilization by thiol-maleimido coupling reaction. During these processes, DNA was not exposed to any organic solvents and reagents, since the DNA immobilization was the final step for the array fabrication. Therefore, this modification enabled us to fabricate an array of delicate molecules, such as double-stranded DNAs.
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In our effort to establish a standard method for fabrication of a double-stranded DNA array, we also examined the usage of heterobifunctional crosslinkers. Two heterobifunctional crosslinkers, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxalate (SSMCC) and N-hydroxysuccinimide-PEG maleimido MW 3400 (NHS-PEG-MAL), were tested for the immobilization of 5'-thiol modified oligonucleotides (Fig. 2). SSMCC provides a hydrophobic short linker, while NHS-PEG-MAL possesses a hydrophilic and flexible spacer region.
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Among various components of the SPR binding buffer, we found that the salt concentration had the greatest influence on the occurrence of nonspecific binding of transcription factors. We measured the SPR signals after continuous MafG application for 30 min at different sodium chloride concentrations from 150 mM to 300 mM (Table 2). When the sodium chloride concentration is 150 mM or less, the nonspecific binding was observed at the blank spot and PEG background on the chip, judged from the smaller value of S1/N ratio (Table 2 and data not shown). In contrast, when 300 mM sodium chloride was applied, S1 value (Table 2) became low, suggesting that the specific binding was inhibited. We therefore utilized intermediate concentration of sodium chloride, i.e. 200 mM, for the MafG analysis.
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Interaction between MafG and MARE-related sequences examined on one chip
We then evaluated a double-stranded DNA array fabricated by the new immobilization method. Each pair of complementary oligonucleotides was first annealed and then immobilized on the surface of a gold chip. The double-stranded oligonucleotides were spotted by an automated spotter and immobilized through the thiol-modified 5'-protruding end on the gold surface via NHS-PEG-MAL. We chose four MARE-related sequences found in the regulatory regions of four endogenous genes (Table 1), including human NQO1 (hNQO1m MARE; Venugopal & Jaiswal 1996), mouse GSTy (mGSTY MARE; Itoh et al. 1997), human ß-globin gene (hBglHS4 MARE; Stamatoyannopoulos et al. 1995), and human rhodopsin gene (hOPSIN MARE; Kumar et al. 1996). The importance of these MAREs has been examined functionally in co-transfection-transactivation analyses. The human NQO1 MARE has an altered flanking region on one side, which is similar to human ß-globin MARE. To examine MAREs encompassing various categories, we modified flanking sequence of human NQO1 MARE so that the crucial ‘G’ in the flanking region is conserved symmetrically (Table 1). For this reason, we named the DNA as hNQO1m. In addition to these MAREs, both MARE25 and MARE23 were spotted as a positive and negative control, respectively.
The chip with the immobilized double-stranded DNAs was placed to the SPR imaging instrument, and 125 nM of MafG homodimer was applied for the DNA-protein association analysis. The SPR signal profiles of association and dissociation were observed for 1800 s with MafG-containing buffer and for the following 1200 s with the blank buffer, respectively. These results are shown in the conventional binding curves in Fig. 4A. To visualize the results more effectively, we also calculated the signals utilizing Scion Image software and the results are shown in the form of SPR difference image in Fig. 4B. The association and dissociation rate constants were calculated from the curve profiles (Fig. 4, n = 3) and summarized in Table 3. Although ka and kd values obtained from the single-stranded DNA array were slightly lower than those obtained from the double-stranded array, KD values of MARE25 were almost the same in the two distinct arrays (see above and Fig. 3B). A reason for the difference in ka and kd values is unknown, but the similar KD values suggest that the double-stranded DNA was properly immobilized on the surface without being denatured. The SPR difference image (Fig. 4B) was calculated from the SPR signals before and after exposure to MafG, and these signals represent that MafG properly binds to the spots. We interpret that MafG binds to MAREs specifically, since the signals on PEG background and a blank spot, where NHS-PEG-MAL is immobilized, are negligible. These results thus demonstrate successful establishment of a modified surface immobilization procedure for a double-stranded DNA array fabrication.
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Comparison of KD values obtained from SPR imaging technique and from GMSA
In order to evaluate validity of the SPR imaging technique, the KD values obtained from the SPR binding analyses were compared to those from GMSA. The KD values determined by GMSA for MARE25, hOPSIN MARE and hNQO1m MARE were ranged in the magnitude of 10–7 (Fig. 5, lanes 1–7 and 15–28). MARE25, hOPSIN MARE and hNQO1m MARE showed high affinities, and the highest was MARE25. On the contrary, weak MafG binding to mGSTY MARE was observed, albeit it was not enough for the KD value determination (Fig. 5, lanes 29–35). No shifted bands were observed for MARE23 and hBglHS4 MARE (Fig. 5, lanes 8–14 and 36–42, respectively). These results are summarized in Table 3. Although KD values calculated from the SPR signals are ranged in the magnitude of 10–9, which are much smaller than those determined by GMSA, the comparative affinities obtained from these two distinct methods were very similar to each other. The affinity of MafG to MARE25 is the highest, and those to hOPSIN MARE and to hNQO1m MARE are intermediate. Interactions between MafG and mGSTY MARE, hBglHS4 MARE and MARE23 are not strong enough for the calculation of kinetic values.
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Discussion |
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In the classic SPR studies on the DNA-protein interaction, biotin-streptavidin chemistry was usually adopted (Seimiya & Kurosawa 1996; Galio et al. 1997; Suzuki et al. 1998; Oda et al. 1999). In this case, biotin-terminated oligonucleotides are usually attached on streptavidin-modified surface. This method, however, has an inherent problem upon applying for the array fabrication, as cross-contamination among the spots may happen due to a strong and quick binding reaction between biotin and streptavidin. In order to perform a comprehensive quantification of binding affinities of transcription factors to various suboptimal sequences, it is a prerequisite to fabricate a double-stranded DNA array composed of multiple sequences that are very similar to one another. To this end, we adopted a method that allows immobilization of pre-annealed double-stranded DNAs on the chip, which can prevent mismatched hybridization and attain complete pairing between complementary DNAs. Indeed, we proved in this study that correct DNA immobilization was accomplished without contaminating spots in this procedure.
Another contrivance in this procedure is the choice of NHS-PEG-MAL as a heterobifunctional crosslinker. It should be noted that the interaction profiles between MafG and MAREs obtained with NHS-PEG-MAL-immobilized array show high level consistency with those observed in GMSA. This is in stark contrast to the results with SSMCC-immobilized array, as the latter results were not consistent with the GMSA data at all. We speculate that the flexible and hydrophilic linker provided by NHS-PEG-MAL might facilitate specific binding and prevent nonspecific adsorption of MafG by allowing the higher DNA mobility. We suppose that the PEG spacer should be generally effective to avoid non-specific interactions of a test protein to the spot background regions. On the contrary, the salt concentration required for suppression of nonspecific binding must be determined for each protein.
Interactions between transcription factors and DNAs have been investigated by several methods including GMSA (Affolter et al. 1990; Yamamoto et al. 1990), filter-binding assay (Tanikawa et al. 1993) and SPR (Seimiya & Kurosawa 1996; Galio et al. 1997; Oda et al. 1999). The KD values, determined by these methods, are ranging from 10–7 to 10–10 and, especially, those obtained by SPR are from 10–7 to 10–9. One report compared KD values calculated by GMSA and SPR, and showed that the values are almost similar to one another ranged in the magnitude of 10–9 (Suzuki et al. 1998). In our case, KD values obtained by SPR ranged around 10–9, while those obtained by GMSA ranged around 10–7 (see Table 3). While the reason for this discrepancy is unclear at present, the following two differences in the measurement conditions may be pertinent.
First, DNA mobility is different from each other in the two measurements. Whereas DNAs are immobilized on a surface for SPR analysis, they are free in the solution in GMSA. Second, optimal salt concentrations are different from each other. Buffers with higher salt concentrations (more than 150 mM) are required for the SPR measurement to avoid the nonspecific adsorption of proteins on to the chip surface or immobilized DNA. On the contrary, GMSA buffer usually contains salts less than 75 mM, and nonspecific competitor DNA is typically added to the binding reaction solutions. In fact, we utilized in this study a high salt concentration (200 mM) to suppress nonspecific binding of DNA and MafG in the SPR analysis, whereas nonspecific DNA competitor was used for this purpose in GMSA. The kinetic profiles in the SPR measurement were investigated at various salt concentrations (Seimiya & Kurosawa 1996; Oda et al. 1999), and it was found that the lower salt concentration gives rise to the smaller kd values, and that ka values are usually not affected by the salt concentration. Consistent with the finding, we found that the dissociation of MafG and MARE25 became faster when sodium chloride concentration was as high as 450 mM (data not shown). Although these results do not explain the KD value difference between SPR and GMSA, we still consider that both SPR and GMSA measurements are valid for quantitative interaction analysis, since there is a very good correlation between the KD values of several MAREs obtained by SPR and GMSA.
All three small Maf family proteins are known to form either homodimer or heterodimer with other bZip superfamily members, including the CNC and Bach family members, and bind to MARE (Motohashi et al. 2002). These partner molecules cannot bind to MARE as a monomer or homodimer, so that small Mafs confer the DNA-binding ability on partner proteins and enable them to execute various activities directed by their functional domains through the heterodimerization. Since many of these Maf-based dimers exist in cells simultaneously, it seems very difficult to identify the primary Maf molecule or to evaluate the contribution of each dimer molecule to the gene regulation through a specific MARE in the regulatory region. One simple hypothesis is to assume that the most abundant dimer molecule in the nuclei may bind dominantly to MAREs, which leads to the notion that the balance between positive and negative regulators interacting to MAREs determines the eventual transcriptional activity. In fact, by adopting megakaryocytic gene regulation directed by NF-E2 p45 and small Maf proteins, we showed that quantitative alteration enables small Maf proteins to direct both active and repressive transcription (Motohashi et al. 2000). In the absence of small Mafs, p45 does not bind to DNA, so MARE-dependent transcription cannot be activated. In the excess of small Mafs, transcriptionally inactive small Maf homodimer occupies MAREs and represses the transcription. Only at the optimal concentration of p45 and small Maf, the maximum level of transcriptional activation is achieved by p45/small Maf heterodimer.
Recent data suggest that the qualitative difference may also be important for the interaction of MAREs and Maf-based dimers. When we examined mafG::mafK compound null mutant mice, mutant animals displayed quite selective MARE-dependent transcriptional abnormality (Katsuoka et al. 2003). In the mice, heme oxygenase-1 (HO-1) mRNA level was markedly increased. Similarly, Bach1-null mutant mice exhibit selective increase of HO-1 mRNA level (Sun et al. 2002). However, no apparent influence of small Maf decrease is observed for other MARE-dependent genes (our unpublished observation), indicating that small Maf and Bach1 make a major contribution to HO-1 gene regulation.
Considering this situation, comprehensive evaluations become crucial for Maf-based dimer interactions with various MARE-related sequences. In this regard, quite recently the interactions between various bZip superfamily proteins were investigated in silico with glass slide-based protein arrays and fluorescent-labelled protein probes (Newman & Keating 2003). We analysed in this study the DNA-protein interaction. Our system enables to examine all possible variations in MAREs quantitatively by using a couple of gold chips or more, since simultaneous detection of 96 samples is technically feasible on one chip. We adopted MafG homodimer as our initial trial of this SPR-array technology, since it is the simple system composed of a single molecule. Obviously, next important analysis will be comparing binding profiles of MafG homodimer, Bach1/small Maf heterodimer, and the other Maf-based heterodimer molecules on one chip. We surmise that Bach1/small Maf heterodimer may have the strongest preference toward HO-1 MAREs.
In spite of discrepancy in absolute KD values calculated from SPR and GMSA, there is a very good correlation between the two results in general. Since the SPR imaging is a powerful technique for the large-scale high-throughput analysis, we propose that the SPR imaging technique would be suitable for examining the general binding preference of a transcription factor. We also propose that each specific interaction should be evaluated with the combination of multiple strategies, including SPR and GMSA for in vitro binding and reporter gene assay for in vivo binding.
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Experimental procedures |
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The chemicals 8-amino-1-octanethiol, hydrochloride (8-AOT, Dojindo Laboratories), thiol terminated methoxypoly(ethylene glycol) MW 5000 (PEG-thiol, NOF), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxalate (SSMCC, Pierce), and N-hydroxysuccinimide-PEG maleimido MW 3400 (NHS-PEG-MAL, Shearwater), were all used as received.
Preparation of oligonucleotide DNAs
The oligonucleotides for covalent immobilization on the surface were designed as 5'-HS-(T)15-CGGAAT(N)13TTACTC-3', and synthesized at Hokkaido System Science or Sigma Genosys with the thiol group protected. The 15-base thymine stretch with a thiol group on the 5'-end was added to the test sequence, which is composed of 13 variable sequence flanked by 6 fixed bases on both sides. 5'- and 3'-fixed sequences were CGGAAT and TTACTC, respectively. Table 1 outlines the various sequences we used in this study. The thiol group on the 5'-end of the oligonucleotides were deprotected, and they were purified by gel filtration with NAP-5 Columns (Amersham Biosciences) as described by Sigma Genosys. The complementary oligonucleotides were synthesized against the variable region with 6-base fixed regions on both sides. The double-stranded DNAs were prepared by annealing longer and shorter complementary DNAs with and without 5'-thiol group, respectively. 25 µM of 5'-thiolated strand and 100 µM of its complementary strand were annealed in the 5x SSC solution (75 mM sodium citrate, 750 mM NaCl; pH 7.0). The solution was heated to 94 °C for 5 min, and quenched to 4 °C for 15 min, then incubated at 37 °C for 3 h, to complete the annealing.
MafG protein preparation
MafG containing EHR and bZip motif, but lacking C-terminal 39 amino acids, was expressed in Escherichia coli as a His6-tagged protein. The crude bacterial lysate was sequentially purified with SP sepharose (Pharmacia) and ProBond resin (Invitrogen). The recombinant protein was then cleaved with thrombin (Calbiochem) and further purified using SP sepharose. 200 µL of 125 nM MafG homodimer solution was used in one experiment.
Fabrication of DNA arrays
The covalently immobilized DNA array with PEG background was obtained by the following procedure. Gold layer (45 nm) with thin chromium underlayer (3 nm) on SF10 glass slide (Schott) were used for SPR imaging measurement. The gold slide was immersed in a PEG-thiol solution (1 mM in 1 : 6 H2O: Ethanol) for at least 3 h to form PEG layer on the surface. This slide was patterned at 40 mW/cm2 for 2 h with chromium quartz mask, which had 96 square holes of 500 µm, by UV light source, which was generated from a 500 W super high-pressure mercury lamp (Ushio, Tokyo). After the surface was rinsed with water and ethanol, the slide was soaked in 1 mM ethanolic solution of 8-AOT for 1 h. This resulted in 96 amino-functionalized 500 µm squares with PEG background. Thiol-reactive maleimido-modified surface was created with 1 mM solution of heterobifunctional crosslinker SSMCC or NHS-PEG-MAL in phosphate buffer (20 mM phosphate; pH 7.0 and 100 mM NaCl). 10 nL drop of 10 µM 5'-thiol-terminated DNA in phosphate buffer was delivered automatically on the patterned surface by using an automated spotter (Toyobo, Osaka), and the reaction was carried out for overnight. Then the surface was rinsed with phosphate buffer and 5x SSC solution containing 0.1% SDS.
SPR imaging analysis
The DNA array was placed immediately in the SPR imaging instrument (Toyobo). The SPR signals were obtained in the SPR buffer (20 mM HEPES (pH 7.9), 200 mM NaCl, 4 mM MgCl2, 1 mM EDTA, and 100 µg/mL BSA). The SPR buffer and the sample in the same buffer were applied to the array surface with 100 µl/min. The SPR image and signal data were collected with MultiSPRinter Analysis program (Toyobo). The SPR difference image was constructed by using Scion Image (Scion, MD USA). The kinetic values were calculated with the program based on the simple reversible reaction model (George et al. 1995).
Gel mobility shift assays
Gel mobility shift assays were performed as previously described (Kataoka et al. 1994a). The same oligonucleotides with those used in SPR detection, which are composed of a 13 bp-variable sequence flanked by 6 bp-fixed regions on both sides, were end labelled with 
-32P-ATP for generating probes. MafG protein was incubated with probes in the gel shift buffer (20 mM HEPES (pH 7.9), 20 mM KCl, 5 mM dithiothreitol, 4 mM MgCl2, 1 mM EDTA, 100 µg/mL BSA and 400 µg/mL poly(dIdC)) at 37 °C for 30 min. The resulting mixture was subjected to native polyacrylamide gel electrophoresis and visualized by autoradiography. The KD values were determined as described (Azam & Ishihama 1999) on the basis of the results obtained using protein concentration from 0 to 360 µM.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: masi{at}tara.tsukuba.ac.jp
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References |
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Received: 17 October 2003
Accepted: 26 November 2003
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