Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic b-amylase from Calystegia sepium (hedge bindweed) rhizomes Els J. M. Van Damme 1 , Jialiang Hu 1 , Annick Barre 2 , Bettina Hause 3 , Geert Baggerman 4 , Pierre Rouge ´ 2 and Willy J. Peumans 1 1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Leuven, Belgium; 2 Institut de Pharmacologie et Biologie Structurale, Unite ´ Mixte de Recherche Centre National de la Recherche Scientifique 5089, Toulouse, France; 3 Institute of Plant Biochemistry, Halle, Germany; 4 Laboratory of Developmental Physiology and Molecular Biology, Katholieke Universiteit Leuven, Leuven, Belgium An abundant catalytically active b-amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium ). Biochemical analysis of the purified protein, molecular modeling, and cloning of the correspond- ing gene indicated that this enzyme resembles previously characterized plant b-amylases with regard to its amino-acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the b-amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome b-amylase is a cytoplasmic vegetative storage protein. Keywords: b-amylase; Calystegia sepium; hedge bindweed; immunolocalization; vegetative storage protein. Exo-hydrolases catalyzing the release of b-maltose from the nonreducing ends of a-1,4-linked oligo- and polyglucans (also so-called b- or exo-amylases) (EC 3.2.1.2) have been studied for several decades because they are possibly involved in starch metabolism in plants, and play an important role in biotechnological processes whereby starch is converted into simple sugars. In the past, research on b-amylases has been focussed on the abundant b-amylases found in the endosperm of barley (Hordeum vulgare ) and some other cereals [1], soybean (Glycine max ) seeds [2] and sweet potato (Ipomoea batatas) tubers [3]. During the last decade, evidence has accumulated that b-amylases are ubiquitous in flowering plants. Cereals such as barley, wheat (Triticum aestivum), rye (Secale cereale ) and maize (Zea mays ) also contain, besides the classical abundant and highly active endosperm b-amylases, low levels of another so-called ‘tissue-ubiquitous’ form in leaves and roots [1]. b-Amylases have also been identified in roots of alfalfa (Medicago sativa ) and several other forage legumes including sweetclover (Melilotus officinalis ), red clover (Trifolium pratense ), birdsfoot trefoil (Lotus corniculatus ) [4], and in pea (Pisum sativum ) epicotyls [5]. In addition, b-amylases have been identified in species of the families Solanaceae (potato, Solanum tuberosum ) [6] and Brassica- ceae (Arabidopsis thaliana and Streptanthus tortuosus ) [7,8]. Extensive enzymatic studies of several b-amylases unambiguously demonstrated that these enzymes exclu- sively catalyze the release of b-maltose from the nonreducing ends of a-1,4-linked oligo- and polyglucans. Accordingly, b-amylases are believed to be involved in the degradation of starch in the plant and/or a-1,4-linked oligoglucans. Though this presumed role might hold true for some b-amylases, it certainly cannot be extrapolated to all plant b-amylases because (a) some b-amylases occur in tissues that are devoid of starch, (b) many plant b-amylases are spatially separated from their presumed substrate (i.e. starch), and (c) inbred lines of rye lacking the abundant endosperm b-amylase germinate normally [9]. This implies that some b-amylases are not required and even not involved in starch degradation but fulfil another role [10]. It has been proposed, for example, that the abundant b-amylases from cereal endosperm and alfalfa taproots function as seed storage proteins and vegetative storage proteins (VSPs), respectively [1,4]. A major difficulty in confirming the role of b-amylases is the lack of insight in their subcellular location. According to some reports, b-amylase is an extrachloroplastic protein restricted to the cytoplasm of spinach cells [11] and A. thaliana leaves [7], which implies that the enzyme does not contribute to the amylolytic activity of the chloroplast. Others, however, presented evidence for a vacuolar location (e.g. in pea and wheat leaf protoplasts) [12]. Indirect evidence based on the absence of a signal peptide from the deduced sequence of all b-amylases cloned thus far suggests that the enzyme is located in the cytoplasm [10]. Although there is evidence that in A. thaliana leaves one particular b-amylase is Correspondence to E. J. M. Van Damme, Katholieke Universiteit Leuven, Laboratory for Phytopathology and Plant Protection, Willem de Croylaan 42, 3001 Leuven, Belgium. Fax: þ 32 16 322976, Tel.: þ 32 16 322379, E-mail: Els.VanDamme@agr.kuleuven.ac.be Enzyme: b-amylase (EC 3.2.1.2). Note: the nucleotide sequence reported in this paper has been submitted to the GenBanke/EMBL Data library under the accession number AF284857. (Received 6 July 2001, revised 5 October 2001, accepted 8 October 2001) Abbreviations: CalsepRRP, Calystegia sepium RNase-related protein; HCA, hydrophobic cluster analysis; VSP, vegetative storage protein; Calsepa, C. sepium agglutinin. Eur. J. Biochem. 268, 6263–6273 (2001) q FEBS 2001 synthesized with a typical N-terminal chloroplast import signalandisefficientlyimportedbyisolatedpea chloroplasts [13], it is still unclear whether plant b-amylases in general are transported from the cytoplasm into another subcellular compartment. A recent study of the predominant proteins in rhizomes of hedge bindweed (Calystegia sepium ) revealed that this vegetative storage tissue accumulates, besides large quantities of a catalytically inactive RNase-related protein [14], substantial amounts of a mannose/maltose-specific lectin [15,16] and a 55-kDa polypeptide with an N-terminal sequence similar to that of typical plant b-amylases. This is an interesting observation because it demonstrates for the first time the simultaneous occurrence in a plant tissue of a lectin with a high affinity for the reaction product of b-amylases. To confirm the possible interaction between the carbohydrate-binding protein and the polysaccharide- degrading enzyme the hedge bindweed b-amylase was purified, characterized and immunolocalized. Our results demonstrate that the enzyme resembles previously described plant b-amylases and is exclusively located in the cytoplasm. The abundance, subcellular location and developmental regulation suggest that the rhizome b-amylase is a cytoplasmic VSP. MATERIALS AND METHODS Plant material Rhizomes of hedge bindweed [C. sepium (L.) R.Br.] were collected in Leuven in winter. Extraction and purification of b-amylase from rhizomes of C. sepium The b-amylase was purified by classical protein purification techniques. Fresh rhizomes (100 g) were cut into small pieces and homogenized in a Waring blender in 1 L of a solution of 0.1% (w/v) ascorbic acid (adjusted to pH 6.5). The homogenate was squeezed through a double layer of cheesecloth and centrifuged at 3000 g for 10 min The supernatant was adjusted to pH 8.7 with 1 M NaOH, centrifuged at 8000 g for 10 min and filtered through filter paper (Whatmann 3 mm). A first purification step was achieved by ion-exchange chromatography. The crude extract was applied on a column (5 £ 5 cm; 100 mL bed volume) of Q Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 m M Tris/HCl (pH 8.7). After loading the extract the column was washed with 1 L of the same Tris buffer and eluted with 300 mL of 0.2 M NaCl in Tris buffer. The resulting partially purified protein fraction was diluted with 5 vol. of Tris buffer and loaded on a column (2.6 £ 15 cm; 75 mL bed volume) of Q Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with Tris buffer. After washing with 200 mL of Tris buffer, proteins were eluted with a linear gradient (500 mL) of increasing NaCl concentration (from 0 to 0.4 M) in Tris buffer. Fractions (10 mL each) were collected and the proteins analysed by SDS/PAGE. All fractions containing predominantly a single polypeptide of < 55 kDa were pooled, adjusted to 1 M ammonium sulfate (by adding the solid salt) and applied on a column (1.6 cm £ 5cm; < 10 mL bed volume) of phenyl–Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 1 M ammonium sulfate. Bound proteins were eluted with 5 mL of 0.1 M Tris/HCl (pH 8.7) and loaded onto a column (2.6 £ 70 cm; < 350 mL bed volume) of Sephacryl 100 equilibrated with KCl/NaCl/P i (1.5 mM KH 2 PO 4 /10 mM Na 2 HPO 4 / 3m M KCl/140 mM NaCl, pH 7.4). The main peak eluting with an apparent molecular mass around 200 kDa was collected, dialysed against appropriate buffers and stored in small aliquots at 2 20 8C until use. Analysis by SDS/PAGE confirmed that the purified protein consisted exclusively of a single 55-kDa polypeptide. Activity assays demonstrated that the protein exhibited b-amylase activity. Analytical methods Purified proteins were analyzed by SDS/PAGE using 12.5–25% (w/v) acrylamide gradient gels as described by Laemmli [17]. The gel was scanned with an AlphaImagere 2200 documentation and analysis system (Alpa Innotech Corporation, San Leandro, CA, USA) to determine the relative concentrations of the major proteins. For N-terminal amino-acid sequencing the proteins were separated by SDS/PAGE and electroblotted onto a poly(vinylidene difluoride) membrane. Polypeptides were excised from the blots and sequenced on an Applied Biosystems model 477 A protein sequencer interfaced with an Applied Biosystems model 120 A on-line analyzer. Isoelectric focusing was performed on the Pharmacia Phast System using polyacrylamide gels (5% T/3% C) containing ampholytes (pH 3–9) (Amersham Pharmacia Biotech). The proteins were detected with silver staining (Pharmacia LKB Biotechnology, Development Technique File no. 210) and isoelectric focusing standards (pI 3.5–9.3) were used. Total neutral sugar content of the purified protein was determined by the phenol/H 2 SO 4 method [18], with D-glucose as standard. For alkylation, 1 mg purified protein was dissolved in 200 mL 0.1 M Tris/HCl (pH 8.7) containing 8 M urea and 10 m M 2-mercaptoethanol. After heating at 60 8C for 10 min iodoacetamide was added to a final concentration of 20 m M and the mixture kept on ice for 30 min The reaction was quenched by the addition of 2-mercaptoethanol (50 m M final concentration) followed by heating at 60 8C for 10 min Mass spectrometry was performed using a MALDI-TOF instrument. Two microliters of a 1.3-mg·mL 21 b-amylase solution were mixed with one microliter of a 50-m M solution of a-cyano-4-hydroxycinnamic acid in CH 3 CN/EtOH/ trifluoroacetic acid (50 : 49.9 : 0.1) and applied on the multi sample target. This mixture was air-dried and the target was then introduced in the instrument, a VG Tofspec SE (Micromass, Manchester, UK) equipped with a N2-laser (337 nm). The samples were measured in the linear mode (acceleration voltage 25 kV), the laser energy was reduced until an optimal resolution and signal/noise ratio was obtained. The results of 10–20 shots were averaged to obtain the final spectrum. Enzyme assay The b-amylase activity was determined by different methods. The first method was based on the release of 6264 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001 p-nitrophenol from the specific substrate p-nitrophenyl maltopentaoside (Betamyl reagent from Megazyme, Wicklow, Ireland) [19]. Assays were performed for 10 min at 40 8C in maleate buffer (pH 6.2), and absorbance was measured at 410 nm as described in the manual provided by the manufacturer. This method is highly specific for b-amylases. Moreover, it is a simple and sensitive test but is less suited for kinetic analysis. In a second method the release of maltose residues from starch, amylopectin and amylose (Sigma Chemical Co., St Louis, MO, USA) was measured by the dinitrosalicylic acid method [20]. Enzyme solution (0.2 mL) and 0.2 mL substrate (0.0625–1% starch, amylopectin or amylose) were incubated for 15 s to 3 min at 20 8C in buffer (pH 5). The reaction was stopped by adding 0.4 mL of staining solution [1% dinitrosalicylic acid (w/v) and 30% sodium potassium tartrate (w/v) in 0.4 M NaOH] and heating at 90 8C for 5 min before measuring the absorbance at 540 nm against blanks without enzyme. The dinitrosalicylic acid method is not very specific for b-amylase in that it will also measure a-amylase activity. Moreover, it is time consuming and relatively insensitive. However, the method is very well suited for kinetic analyses of purified b-amylases. In a third method the degradation of starch was determined by the iodine staining method [21]. The reaction was started by adding 100 mL of the enzyme (21.4 mg·mL 21 ) to 500 mL substrate solution [0.0625–1% (w/v)] and 400 mL inhibitor solution (0.2 M glucose or maltose, or 3.125 mM cyclohexa- amylose), in 20 m M sodium-acetate buffer (pH 5.0). After incubation at 20 8C for 15 s to 2.5 min the reaction was stopped by adding 0.5 mL 1 M HCl. To each sample 1 mL staining solution (0.2% iodine in 2% potassium iodide) was added, and the mixture diluted to 20 mL before measuring the decrease in absorbance at 700 nm. The iodine staining method also is not very specific for b-amylases and is relatively insensitive. However, the method is less time consuming than the dinitrosalicylic acid method and is not affected by the inhibitors of the enzymatic activity. Therefore the iodine staining method is well suited for extensive kinetic analyses of purified b-amylases. Stability tests The heat stability of the enzyme was determined by heating a solution of the purified protein (0.1 mg·mL 21 in 0.1 M phosphate buffer pH 6.2) at 20 – 100 8C(with108C increments) for 10 min. Afterwards, activity of the enzyme was determined using the Betamyl b-amylase test reagent (Megazyme, Wicklow, Ireland). To determine the pH stability of the b-amylase, aliquots of a solution of the purified protein (4.06 mg·mL 21 in water) were adjusted to different pH values in a range between 2 and 12, and incubated for 1 h at 25 8C. Then 0.1 vol. of a solution of 0.5 M sodium acetate (pH 5.0) was added and the activity of the enzyme was measured by the dinitrosalicylic acid method. Inhibition of the enzyme activity by glucose, maltose and cyclohexaamylose For the study of the enzyme inhibition by glucose, maltose and cyclohexaamylose b-amylase activity was measured using the iodine staining method with soluble starch as a substrate [21]. The inhibition type of glucose, maltose and cyclohexaamylose was determined from Lineweaver–Burk plots, and inhibitor constants were determined from Dixon plots. RNA isolation, construction and screening of cDNA library Total cellular RNA was prepared from the apexes of bindweed rhizomes and poly(A)-rich RNA enriched by chromatography on oligo-deoxythymidine cellulose as described by Van Damme and Peumans [22]. A cDNA library was constructed as described previously [20]. Recombinant clones encoding b-amylase were screened using 32 P-end-labeled degenerate oligonucleotide probes derived from the N-terminal amino-acid sequence of Calystegia b-amylase. In a later stage, cDNA clones encoding b-amylase were used as probes to screen for more cDNA clones. Hybridizations were performed overnight as reported previously [15]. Colonies that produced positive signals were selected and rescreened at low density using the same conditions. Plasmids were isolated from purified single colonies on a miniprep scale using the alkaline lysis method as described by Mierendorf and Pfeffer [23] and sequenced by the dideoxy method [24]. DNA sequences were analysed using programs from PC GENE (Intelli- genetics, Mountain View, CA, USA) and GENEPRO (Riverside Scientific, Seattle, USA). Northern blot analysis RNA electrophoresis was performed according to Maniatis et al. [25]. Approximately 3 mg of poly(A)-rich RNA were denatured in glyoxal and dimethylsulfoxide and separated in a 1.2% (w/v) agarose gel. Following electrophoresis the RNAwastransferredtoImmobilonNmembranes (Millipore, Bedford, USA) and the blot hybridized using a random-primer-labeled cDNA insert or an oligonucleotide probe. Hybridization was performed as reported by Van Damme et al. [26]. An RNA ladder (0.16– 1.77 kb) was used as a marker. PCR amplification of genomic DNA fragments encoding b-amylase DNA was extracted from young leaves of C. sepium using the protocol described by Stewart and Via [27]. The DNA preparation was treated with RNase (Roche Diagnostics GmbH, Mannheim, Germany). The reaction mixture for amplification of genomic DNA sequences contained 10 m M Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 100 mg·L 21 gelatin, 0.4 mM of each dNTP, 2.5 U of Taq polymerase (Roche Molecular Biochemicals, Mannheim, Germany), 500 ng of genomic DNA and 20 mL of the appropriate primer mixtures (20 m M), in a 100-mL reaction volume. The reaction was overlaid with 80 mL of mineral oil. After denaturation of the DNA for 5 min at 95 8C amplification was performed for 30 cycles through a regime of 1 min template denaturation at 92 8C followed by 1 min primer annealing at 55 8C and 3 min primer extension at 72 8C using a Perkin Elmer DNA Thermal Cycler (480). The PCR fragments were purified using Qiaquick PCR Purification kit (Qiagen, Hilden, Germany) and cloned in TOPO q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6265 pCR2.1-TOPO cloning vector using the TOPO cloning kit from Invitrogen (Carlsbad, CA, USA). Preparation of specific antibodies against b-amylase and C. sepium RNase-related protein (CalsepRRP) Polyclonal antibodies were raised against b-amylase and the unglycosylated isoform of CalsepRRP [14]. Male New Zealand white rabbits were injected with 1 mg purified protein dissolved in KCl/NaCl/P i and emulsified in 1 mL of Freund’s complete adjuvant. Five booster injections with 1 mg of purified protein in 1 mL of KCl/NaCl/P i were given at 10-day intervals. Ten days after the final injection, blood was collected from an ear marginal vein. After clotting, the crude serum was prepared by centrifugation (3000 g for 5 min) and processed immediately by affinity chromato- graphy on a column of immobilized b-amylase or CalsepRRP. Coupling of the antigens to the column and purification of the antiserum were performed as described previously [28]. Western blot analysis The specificity of the antisera was analysed by Western blot analysis. Proteins were separated by SDS/PAGE and electroblotted on an Immobilon P membrane. Before immunodetection the free binding sites on the membrane were blocked with 5% BSA in Tris/NaCl/P i (10 mM Tris, 150 m M NaCl, 0.1% Triton X-100, pH 7.6) for 1 h at room temperature. After washing the membrane with Tris/NaCl/P i for 5 min the membrane was consecutively treated with rabbit primary antibody (overnight incubation at room temperature), goat anti-(rabbit IgG) Ig (1 h incubation at room temperature) and peroxidase–anti-peroxidase com- plex (1 h incubation at room temperature). After every treatment the membrane was washed three times with Tris/NaCl/P i for 5 min. Prior to the immunodetection the membrane was washed for 5 min with 0.1 M Tris/HCl, pH 7.6. The peroxidase reaction was carried out in a fresh solution of 0.1 M Tris/HCl pH 7.6 containing 0.7 mM 3,3 0 - diaminobenzidine tetrahydrochloride and 0.01% (v/v) H 2 O 2 . The reaction was stopped by washing the membrane in distilled water. Immunocytochemistry Small pieces of fresh C. sepium rhizomes were fixed with 4% paraformaldehyde/0.1% Triton X-100 in KCl/NaCl/P i , embedded in poly(ethylene glycol) and cut as described previously [29]. Cross-sections (2 mm thick) were immuno- labeled by incubation with purified primary IgG raised against b-amylase or CalsepRRP (diluted 1 : 250 in KCl/ NaCl/P i containing 5% BSA and 1 mg·mL 21 goat IgG) followed by goat anti-(rabbit IgG) Ig conjugated with BODIPY (Molecular Probes, Eugene, OR). After immuno- labeling, sections were mounted in citifluor/glycerol. Control experiments were performed by omitting the primary antibody. The fluorescence of immunolabeled b-amylase and CalsepRRP was visualized with a Zeiss Axioskop epifluorescence microscope using the appropriate filter combination. Micrographs were taken by a CCD camera (Sony, Japan) and processed through the PHOTOSHOP program (Adobe, Seattle, WA, USA). Molecular modelling Multiple amino-acid sequence alignments based on CLUSTAL W [30] were performed with SEQPUP (D.G. Gilbert, Biology Department, Indiana University, Bloomington, IN, USA). The program SEQVU (J. Gardner, The Garvan Institute of Medical Research, Sydney, Australia) was used to compare the amino-acid sequences of the b-amylases. Hydrophobic cluster analysis (HCA) [31,32] was performed to delineate the structurally conserved b sheets and a helices along the amino-acid sequences of the b-amylase from hedge bindweed and the model b-amylase from soybean. HCA plots were generated using the program HCA-Plot2 (Doriane, Paris, France). Molecular modeling of the b-amylase from C. sepium was carried out on a Silicon Graphics O2 R10000 workstation, using the programs INSIGHT II, HOMOLOGY AND DISCOVER (Molecular Simulations, San Diego CA, USA). The atomic coordinates of the soybean b-amylase (code 1BYA) [33] were taken from the RCSB Protein Data Bank (http://www.rcsb.org/pdb) to build the three- dimensional model of the C. sepium b-amylase. Energy minimization and relaxation of the loop regions was carried out by several cycles of steepest descent and conjugate gradient using the CVFF forcefield of DISCOVER. Steric conflicts resulting from the replacement or the deletion of some residues in the C. sepium b-amylase were corrected during the model building procedure using the rotamer library [34] and the search algorithm implemented in the HOMOLOGY program [35] to maintain proper side chain orientation. The program TURBOFRODO (Bio-Graphics, Marseille, France) was used to calculate the Ramachandran plot and perform the superposition of the models. Electrostatic potentials were calculated and displayed with GRASP using the PARSE3 parameters [36]. The solvent probe radius used for molecular surfaces was 1.4 A ˚ and a standard 2.0 A ˚ Stern layer was used to exclude ions from the molecular surface [37]. The inner and outer dielectric constants applied to the protein and the solvent were, respectively, fixed at 4.0 and 80.0, and the calculations were performed keeping a salt concentration of 0.14 M NaCl. No even distribution of the net negative charge of the carboxylic group of negatively charged residues was performed between their two oxygen atoms prior to the calculations. The surfaces occupied by negatively charged (Asp, Glu) residues on the solvent accessible surfaces of the modelled amylase were calculated using the GRASP facilities. RESULTS Purification and partial characterization of the b-amylase from C. sepium rhizomes SDS/PAGE of clarified homogenates from resting hedge bindweed rhizomes revealed several major polypeptides (Fig. 1A), some of which have been identified previously. The 15-kDa polypeptide (< 30% of the total protein) corresponds to the subunits of the mannose-binding C. sepium agglutinin (also called Calsepa) [15,16] whereas the 26 to 28-kDa polypeptides (together < 35% of the total protein) represent the unglycosylated and glycosylated form of the so-called CalsepRRP [14]. N-terminal sequencing of the 55-kDa polypeptide (< 10% of the total protein) yielded 6266 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001 a single sequence APIPGVMPMGNYVPVYVMLP with a high degree of identity (85%) to the N-terminus of the b-amylase from sweet potato (Ipomoea batatas ). Therefore this polypeptide was tentatively identified as a b-amylase. Subsequently the 55-kDa C. sepium protein was isolated and tested for b-amylase activity. The C. sepium b-amylase was purified using a combi- nation of conventional protein purification techniques. Analysis of a reduced sample of the final preparation by SDS/PAGE yielded a single polypeptide band of < 55 kDa (Fig. 1A). The unreduced protein also yielded a major band of 55 kDa but exhibited an additional minor band of slightly higher molecular mass (< 65 kDa). To check the possible presence of two different polypeptides the protein was analyzed by mass spectrometry. Thereby, a single peak of 56 068 Da was detected with no sign for the presence of a higher M r form. No minor band of 65 kDa could be detected in an alkylated sample of the protein (Fig. 1A), which indicates that this 65-kDa polypeptide appearing in the electropherogram of the unreduced protein is an artifact due to the formation of an intramolecular disulfide bridge after unfolding of the polypeptide in the presence of SDS. Native C. sepium b-amylase eluted as a symmetrical peak with an apparent M r of < 200 kDa upon gel filtration chromatog- raphy on a Superose 12 column (results not shown), indicating that it is a homotetrameric protein. Isoelectric focusing of the purified b-amylase yielded a single band with an isoelectric point of < 4.8 (Fig. 1B). No carbo- hydrate could be detected in the pure protein using the phenol/sulfuric acid method suggesting that the C. sepium b-amylase is not glycosylated. Fig. 2. Lineweaver–Burk plots. (A) Lineweaver–Burk plots of the activity of C. sepium b-amylase on starch, amylose and amylopectin. The activity was assayed in 20 m M, pH 5.0 acetate buffer at 20 8C using iodine staining method. The enzyme concentration for activity tests on starch, amylose and amylopectin was 2.588 mg·mL 21 , 2.143 mg·mL 21 and 2.679 mg·mL 21 , respectively. The substrate concentration ranged between 0.025 and 0.5% (w/v). (B) Lineweaver–Burk plots of the inhibition of C. sepium b-amylase by glucose, maltose and cyclo- hexaamylose. The activity was assayed using soluble starch as substrate in 20 m M, acetate buffer pH 5.0 at 20 8C. The concentration of enzyme was 2.14 mg·mL 21 . Concentrations of glucose, maltose and cyclo- hexaamylose were 80 m M,80mM and 1.25 mM, respectively. The substrate concentration ranged between 0.025 and 0.5% (w/v). Fig. 1. SDS/PAGE and isoelectric focusing. (A) SDS/PAGE of a clarified homogenate from C. sepium rhizomes and purified b-amylase. Samples were loaded as follows: lane 1, 100 mL total extract from Calystegia rhizomes; lanes 2–5, 20 mg purified b-amylase. The major protein bands in crude extract (lane 1) represent the b-amylase (A), the glycosylated and unglycosylated RNase-related protein (R) and the lectin Calsepa (L). Protein samples in lanes 3 and 5 were alkylated. The samples in lanes 1–3 were treated with b-mercaptoethanol; the protein in lanes 4–5 was not reduced. Molecular mass reference proteins (lane R) were lysozyme (14 kDa), soybean trypsin inhibitor (20 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and phosphorylase b (94 kDa). (B) Isoelectric focusing of b-amylase from C. sepium rhizomes. Samples were loaded as follows: Lane 1, purified b-amylase from C. sepium rhizomes and lane 2, soybean trypsin inhibitor (pI, 4.55). pI markers (lane M) were amylglucosidase (3.50), trypsin inhibitor (4.55), b-lactoglobulin A (5.20), carbonic anhydrase B (bovine) (5.85), carbonic anhydrase B (human) (6.55), myoglobulin (acidic band) (6.85), myoglobulin (basic band) (7.35), lentil lectin (acidic) (8.15), lentil lectin (middle) (8.45) lentil lectin (basic) (8.65) and trypsinogen (9.30). q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6267 Enzymatic activity of b-amylase from hedge bindweed To check the enzymatic activity of the presumed b-amylase from C. sepium rhizomes three different methods were used. The dinitrosalicylic acid method and iodine staining method, revealed that the purified protein shows activity towards starch, amylopectin and amylose (Fig. 2A), starch and amylopectin being a better substrate than amylose. As these two methods are not specific for b-amylase (but also detect a-amylase activity) additional assays were performed with the Betamyl b-amylase test reagent from Megazyme (which uses p-nitrophenyl maltopentaoside as a substrate and is highly specific for plant b-amylases). In this assay the protein exhibited a high activity, which implies that the purified C. sepium protein is an active b-amylase. The C. sepium b-amylase was active in a pH range from 3 to 7 with an optimum near pH 4.8. Stability tests indicated that the enzyme was heat stable up to 60 8C but was completely inactivated upon heating for 10 min at 70 8C. The enzyme is stable in a pH range between 3 and 11. Incubation at pH values below 3 and above 12 irreversibly inactivated the protein. Hydrolysis of the Betamyl b-amylase test reagent from Megazyme was strongly inhibited by glucose and maltose. At a concentration of 125 m M both sugars reduced the activity of the b-amylase with 87.5%. Mannose caused a 6% reduction of the activity when added at a final concentration of 125 m M. In contrast, lactose did not inhibit the enzyme even when the concentration was increased to 250 m M. The inhibition of the enzyme activity by glucose, maltose and cyclohexa- amylose was studied in more detail using the iodine staining method. Glucose behaved as a mixed type inhibitor whereas maltose and cyclohexaamylose behaved as competitive inhibitors of the Calystegia b-amylase (Fig. 2B). Glucose was only a weak inhibitor (K i ¼ 262 mM) when compared to maltose (K i ¼ 11.7 mM) and cyclohexaamylose (K i ¼ 0.36 mM). Molecular cloning of the C. sepium b-amylase Screening of a cDNA library constructed with poly(A)-rich RNA from rhizome apexes using a synthetic oligonucleotide derived from the amino-acid sequence of the C. sepium b-amylase yielded multiple positive clones of < 2Kb. Sequence analysis of Calsepam1 revealed that this clone contains an ORF of 499 amino acids with one putative initiation codon at position 2 of the deduced amino-acid sequence (Fig. 3). Translation starting with this methionine residue results in a protein of 498 amino acids with a calculated molecular mass of 56 204 Da. The deduced amino-acid sequence of Calsepam1 revealed a sequence identical to the N-terminal sequence of the protein (residues A2–P23) preceded by a methionine residue, suggesting that the N-terminal methionine residue is removed from the primary translation product. The apparent lack of a signal peptide further suggests that the b-amylase is localized in the cytoplasm. Removal of the methionine residue results in a protein of 497 amino acids with a calculated molecular mass of 56 073 Da and an isoelectric point of 4.81. A search in GenBank revealed 86% and 67% sequence identity between the C. sepium b-amylase and the b-amylase from sweet potato and soybean, respectively. Northern blot analysis Northern blot analysis was performed to determine the total length of the mRNA encoding the b-amylase. Hybridization of the blot using the random primer labeled cDNA clone encoding C. sepium b-amylase yielded one band of < 2.0 Kb (results not shown) and is consistent with the length of the cDNA clones which were analyzed. Analysis of genomic fragments encoding b-amylase PCR amplification of genomic DNA fragments encoding b-amylase yielded PCR products of < 2800 bp. Sequence analysis revealed a sequence identical to the sequence of the cDNA but split into seven exons by six intron sequences (Fig. 3). All introns were marked by GT and AG dinucleotides at their 5 0 and 3 0 boundaries, respectively, and were inserted between the third letter of one codon and the first letter of the following codon. Molecular modeling of the C. sepium b-amylase The amino-acid sequence of the b-amylase from C. sepium exhibits 67.5% identity and 76.0% similarity, respectively, with soybean b-amylase (Fig. 3). As the HCA plots of both proteins are very similar, the structurally conserved regions (a helices and b sheets) are readily recognized (results not shown). Due to these structural homologies, a fairly accurate three-dimensional model could be built for the b-amylase from C. sepium using the X-ray coordinates of the soybean b-amylase (Fig. 4). According to the Ramachandran plot of this model the f and c angles of most of the residues are in the allowed regions of low energy, except for Arg421 (f, 888; c, 1318). It should be mentioned, however, that in the soybean b-amylase also this residue is located in a disallowed region. As shown in Fig. 4, the model of the b-amylase from C. sepium comprises (a) a core built up of a bundle of eight parallel b strands surrounded by eight a helices and thus exhibits a typical (a/b) 8 barrel structure, (b) a smaller globular region consisting of three long loops (connected to the core), and (c) a C-terminus consisting of a long loop of over 50 amino-acid residues with a seven amino-acid residue a-helix (Fig. 4). The C. sepium b-amylase contains six cysteine residues. According to the model no disulfide bridges can be formed because the cysteine residues are too distant from each other. Only four of the cysteine residues of the C. sepium b-amylase (Cys83, Cys96, Cys209 and Cys344) are homologous to those found in soybean b-amylase (Cys82, Cys97, Cys208 and Cys343). On the analogy of the soybean b-amylase, the active site of the C. sepium b-amylase most probably consists of a cleft located between the barrel core and the smaller globular region (Fig. 5). This cleft is centered around glutamic residue Glu187 (Glu188 in soybean b-amylase), which is presumed to be involved in the catalytic activity. Localization of the b-amylase in the cells of the rhizomes The cellular and subcellular localization of the C. sepium b-amylase was studied by an immunocytological technique. Rhizomes embedded in poly(ethylene glycol) were sectioned and immunolabelled with purified antibodies 6268 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001 raised against b-amylase. The specificity of the affinity- purified antibodies against the C. sepium b-amylase was checked by Western blot analysis of a crude rhizome extract. As the antibodies reacted exclusively with the 55-kDa polypeptide corresponding to the b-amylase subunit (results not shown), they can be considered specific. The b-amylase could be detected in the cortex and the pith of rhizomes but not in vascular tissues, pericycle, endodermis and rhizodermis (Fig. 6). In cross-section of cells of the pith and the cortex, the vacuole is the dominant organelle. The cytoplasm is visible only as a thin layer adjacent to the cell wall. As shown in Fig. 6, the b-amylase Fig. 3. Amino-acid sequences. (A) Deduced amino-acid sequence of the b-amylase from C. sepium. As the methionine at position 2 is the first amino acid the residue preceding this methionine is shown in lower case. The sequence corresponding to the N-terminal sequence of the protein is underlined. The arrowheads indicate the positions of the intron sequences. (B) Comparison of the amino-acid sequences of b-amylase from C. sepium (this work) with those from Glycine max (GenBank accession No. BAA09462), A. thaliana (accession no. BAA07842), Ipomoea batatas (accession no. BAA02286), Triticum aestivum (accession No. P93594), Zea mays (accession no. P55005) and Hordeum vulgare (accession no. BAA04815). Please note that the last 17-amino- acid residues at the C-terminus of H. vulgare b-amylase are not shown. Deletions are indicated by dashes and identical residues are boxed. q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6269 is confined to the cytoplasm (Figs 6B,D). No label for anti- (b-amylase) IgG was detectable within the large vacuoles, which appeared as a dark area in the centre of the cells. To clearly distinguish the cytoplasmic location of the C. sepium b-amylase from that of a noncytoplasmic protein, sections were also immunolabeled with antibodies raised against the major rhizome protein CalsepRRP, which is presumably located in the vacuole [14]. The specificity of the affinity- purified antibodies against CalsepRRP was checked by Western blot analysis of a crude rhizome extract. As the antibodies reacted with the 28- and 26-kDa polypeptides (corresponding to the glycosylated and unglycosylated polypeptides of the RNase related protein) (results not shown), they can be considered specific. The results shown in Fig. 6F confirm the vacuolar location of this RNase- related protein and rule out the possibility that the apparent cytoplasmic location of the b-amylase is due to an artefact. DISCUSSION A major protein of resting rhizomes of C. sepium, which accounts for < 10% of the total protein, has been identified as a b-amylase. The native enzyme is a homotetramer of four identical subunits of 56 068 Da. It is important to note in this context that apart from the b-amylase from sweet potato tubers all other plant b-amylases characterized to date have been described as monomeric proteins (which implies that both monomeric and tetrameric b-amylases are catalytically active). Molecular cloning combined with N-terminal sequencing and MALDI-TOF mass spec- trometry indicate that the mature protein comprises the entire open reading frame of the corresponding gene minus the N-terminal methionine residue, which indicates that the C. sepium b-amylase undergoes, apart from the removal of the N-terminal methionine, no co- or post-translational processing. According to the results of previously reported molecular and structural studies the processing of the b-amylases from sweet potato [3,38] and soybean [39,40] also is restricted to the removal of the N-terminal methionine whereas that of a phloem-specific b-amylase from A. thaliana includes the removal of an N-terminal tetrapeptide [8,41]. Cereal b-amylases also are not co- or post-translationally modified [42]. However, the abundant endosperm-specific cereal b-amylases are ‘activated and released’ during germination by the proteolytic removal of a C-terminal peptide of < 50 amino-acid residues [1,43]. The three-dimensional model of the C. sepium b-amylase strongly resembles that of the soybean [33,39] and sweet potato [38] b-amylases and shares the typical (a/b) 8 barrel core which is common to all other b-amylases of different Fig. 4. Three-dimensional model of Calystegia sepium b-amylase showing the central bundle of eight strands of b sheet (pink coloured arrows, numbered 1–8) surrounded by eight a-helices (coloured green, numbered 1–8). The a helix in violet does not participate in the core (b/a) 8 TIM-barrel structure. The three acidic residues involved in the catalytic activity (Asp102, Glu187 and Glu381) are represented in ball-and-stick. The conserved loop 97–104 (homologous to loop L3 of the soybean b-amylase), which allows the active site to close is coloured cyan (H). N and C refer to the N- and C-terminus of the b-amylase sequence. Cartoon was generated with MOLSCRIPT [51], BOBSCRIPT [52] and RASTER3D [53]. Fig. 5. Molecular surface of the modelled C. sepium b-amylase showing the surface area (black) occupied by the three acidic residues Asp102 (red), Glu187 (blue) and Glu381 (green) located in the central cleft of the (b/a) 8 TIM-barrel. Most of the exposed surface of residue Asp102 is poorly visible as it is located inside the cleft whose entry appears as a hole in the centre of the model. The model is similarly oriented as in Fig. 4. All the calculations and displays were performed with GRASP [36]. 6270 E. J. M. Van Damme et al. (Eur. J. Biochem. 268) q FEBS 2001 origins [44]. According to both structural [33,39] and functional data [45–47], four amino-acid residues (Asp101, Glu186, Glu345 and Glu380) located in a cleft occurring between the (a/b) 8 barrel core and the smaller globular region play a key role in the catalytic activity of the soybean b-amylase. These four residues are conserved in all other plant b-amylases including the C. sepium b-amylase. Studies of the crystal structure of recombinant soybean b-amylase complexed to b-cyclodextrin demonstrated the role of Glu186 and Glu380 as catalytic residues [40]. It is important to note in this respect that the distance of 7.89 A ˚ between Glu183 (homologous to Glu186 of soybean b-amylase) and Glu381 (homologous to Glu380 of soybean b-amylase) of the C. sepium b-amylase, fits the inverting hydrolytic mechanism of b-amylases. Binding of maltose or maltal ligands to the active site of soybean b-amylase induces a local conformational change of a loop segment (L3) of eight residues (96–103) located in the smaller globular region. This conformational change is required to close the active site of the enzyme [40,48] and allow the reaction to take place. Once the reaction is finished a new conformational change is required to bring the loop into the open position for subsequent release of the reaction product. Due to the importance of the conformational changes the loop segment L3 is highly conserved in all b-amylases from plants and microorganisms (e.g. 97Gly-Gly-Asn-Val-Gly- Asp-Ala-Val104 of Calystegia b-amylase and 96Gly-Gly- Asn-Val-Gly-Asp-Ile-Val103 of the soybean b-amylase). Docking experiments with maltose and maltose derivatives further suggested that the movement of this mobile flap significantly increased the intermolecular binding potential and thus favours the interaction with the ligand [49]. Immunolocalization studies of the C. sepium b-amylase provided for the first time unequivocal evidence for the exclusive cytoplasmic location of a plant b-amylase. Our results confirm the presumed cytoplasmic location b-amy- lases proposed on the basis of cell fractionation studies with spinach [11] and Arabidopsis [7] leaves but can not be reconciled with the previously proposed vacuolar location of b-amylase in pea and wheat leaf protoplasts [12]. In contrast to the cytoplasmic b-amylase, the major storage protein of the hedge bindweed rhizome (CalsepRRP) [14] is clearly located in the vacuole. This particular vacuolar location of CalsepRRP not only serves as a good endogenous control for the cytoplasmic location of the C. sepium b-amylase but also demonstrates that cells of C. sepium rhizomes accumulate large quantities of proteins in both the vacuole and the cytoplasm. The cytoplasmic location of the C. sepium b-amylase implies that the enzyme has no access to its natural substrate because maltodextrins are believed to accumulate in the vacuole [5]. So the question remains why hedge bindweed rhizomes accumulate large quantities of an enzyme with no apparent function. This question applies also to all other abundant plant b-amylases, to which for various reasons no clear role can be attributed. It has been suggested at several occasions that highly expressed b-amylases may act as storage proteins. For example, the absence of a specific function and storage protein-like behaviour of the abundant cereal endosperm b-amylases [1] combined with the observation that mutant lines of barley and rye, which lack the endosperm b-amylase, germinate normally [9,43] point towards a storage role. A similar role has been proposed for the abundant b-amylase in taproots of alfalfa, which is believed to fulfil a storage function in the roots of this perennial legume and accordingly is considered a typical VSP [4]. The abundance and apparent lack of a specific function suggest that the C. sepium b-amylase also can be Fig. 6. Immunolocalization of the C. sepium b-amylase in rhizomes of C. sepium. Cross sections were labelled with a purified polyclonal antibody raised against the C. sepium b-amylase (A, B, D) or a purified polyclonal antibody raised against CalsepRRP (F) followed by a fluorescence labelled secondary antibody. Immunodecorated b-amylase and CalsepRRP are visible by the green fluorescence. (A) Overview of a cross section of a rhizome labelled with anti-(b-amylase) IgG. Note the label restricted to the cortex and the pith whereas vascular tissues, pericycle, endodermis and rhizodermis do not exhibit label. (B) Detail of (A) showing part of the cortex. All cortex cells are labelled. (C) Section concomitant to the section shown in (B) without treatment with the first antibodies. The weak fluorescence in the vascular tissues is due to the autofluorescence of cell walls containing phenolic compounds (A) concomitant section to (B) is shown. (D) Enlargement of (B) to visualize the subcellular localization of the Calystegia sepium b-amylase within cortex cells. Note the label clearly visible within the thin cytoplasmic seam only. Starch grains of amyloplasts appear as black dots. (E) DIC image of (D) to visualize starch grains. (F) Cortex cells of a cross section of a rhizome immunolabelled with anti- (CalsepRRP) IgG. Cells exhibit strong label within the vacuole. Bars represent 100 mm in A–C and 50 mm in D–F, respectively. q FEBS 2001 b-Amylase from hedge bindweed (Eur. J. Biochem. 268) 6271 classified as a VSP (even though the C. sepium rhizome can not be considered a true perennial tissue because it continuously grows at the one end and dies at the other end). If so, the C. sepium and alfalfa taproot b-amylases represent a unique type of VSP because they are located in the cytoplasm whereas all other known VSP are (presumed) vacuolar storage proteins [50]. ACKNOWLEDGEMENTS This work was supported in part by grants from the Research Fund K.U.Leuven (OT/98/17), CNRS and the Conseil re ´ gional de Midi- Pyre ´ ne ´ es (A. B., P. R.), and the Fund for Scientific Research-Flanders (FWO grant G.0223.97). E. V. D. is a Postdoctoral Fellow of this fund. REFERENCES 1. Ziegler, P. (1999) Cereal beta-amylases. J. 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