A tuber lectin from Arisaema jacquemontii Blume with anti-insect and anti-proliferative properties

A tuber lectin from Arisaema jacquemontii Blume belonging to family Araceae was purified by employing a single step affinity chromatography using column of asialofetuin-linked amino activated silica beads and the bound lectin was eluted with 100 mM glycine-HCl buffer pH 2.5. The purified A. jacquemontii lectin (AJL) showed a single protein band with an apparent molecular mass of 13.4 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The native molecular mass of AJL determined by gel filtration on a Biogel P-200 column was 52 kDa and its carbohydrate content was estimated to be 3.40%. Thus AJL is a tetrameric glycoprotein. The purified lectin agglutinated erythrocytes from rabbit but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested except N-acetyl-D-lactosamine having minimal inhibitory sugar concentration (MIC) 25 mM. Among the glycoproteins tested only asialofetuin was found to be inhibitory (MIC125 microg/mL). A single band was obtained in native PAGE at pH 4.5 while PAGE at pH 8.3 showed two bands. Isoelectric focusing of AJL gave multiple bands in the pI range of 4.6-5.5. When incorporated in artificial diet AJL significantly affected the development of Bactrocera cucurbitae (Coquillett) larvae indicating the possibility of using this lectin in a biotechnological strategy for insect management of cucurbits. Larvae fed on artificial diet containing sublethal dose of AJL showed a significant decrease in acid phosphatase and alkaline phosphatase activity while esterase activity markedly increased as compared to larvae fed on diet without lectin. Out of various human cancer cell lines employed in sulphorhodamine B (SRB) assay, this lectin was found to have appreciable inhibitory effect on the in vitro proliferation of HCT-15, HOP-62, SW-620, HT-29, IMR-32, SKOV-3, Colo-205, PC-3, HEP-2 and A-549 cancer cell lines by 82, 77, 73, 70, 41, 41, 37, 29, 21 and 21% respectively.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

A novel mitogenic and antiproliferative lectin from a wild cobra lily, Arisaema flavum

A novel lectin having specificity towards a complex glycoprotein asialofetuin was purified from tubers of Arisaema flavum (Schott.) by affinity chromatography on asialofetuin-linked amino-activated silica beads. A. flavum gave a single peak on HPLC size exclusion and a single band on non-denatured PAGE at pH 4.5. The molecular mass of the lectin, as determined by gel filtration chromatography, was 56 kDa. In SDS-PAGE, pH 8.3, the lectin migrated as a single band of 13.5 kDa, under reducing and non-reducing conditions, indicating the homotetrameric nature. A. flavum lectin (AFL) readily agglutinated rabbit, rat, sheep, goat, and guinea pig erythrocytes but not human ABO blood group erythrocytes even after neuraminidase treatment. This lectin is stable up to 55 degrees C and does not require metal ions for its hemagglutination activity. AFL was completely devoid of sulphur containing amino acids and was rich in aspartic acid and glycine. In Oucterlony's double immunodiffusion, the antisera raised against A. flavum lectin showed distinct lines of identity with those of other araceous lectins. AFL showed potent mitogenic activity towards BALB/c splenocytes and human lymphocytes in comparison to Con A, a well-known plant mitogen. AFL also showed significant in vitro antiproliferative activity towards J774 and P388D1 murine cancer cell lines.     

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

A novel homodimeric lectin from Astragalus mongholicus with antifungal activity

A novel lectin (AMML) was isolated from a Chinese herb, i.e., the roots of Astragalus mongholicus, using a combination of ammonium sulfate fraction and ion exchange chromatographies. The molecular mass of intact AMML was determined to be 66,396 Da by MALDI-TOF mass spectrometry and 61.8 kDa by gel filtration, respectively. AMML was a dimeric protein composed of two identical subunits each with a molecular mass of 29.6 kDa. The lectin was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified lectin hemagglutinated both rabbit and human erythrocytes, and showed preference for blood types O (native) and AB (trypsin-treated). Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives with pronounced preference for lactose (3.13 mM). N-terminal amino acid sequence of AMML was determined as ESGINLQGDATLANN. The optimal pH range for lectin activity was between pH 4.5 and 7.5, and the lectin was active up to 65 degrees C. It also exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmooLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea

From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.     

A novel antiproliferative and antifungal lectin from Amaranthus viridis Linn seeds

A lectin from the seeds of Amaranthus viridis Linn has been purified by affinity chromatography on asialofetuin-linked amino activated silica. Amaranthus viridis lectin (AVL) has a native molecular mass of 67 kDa. It is a homodimer composed of two 36.6 kDa subunits. The lectin gave a single band in non-denaturing PAGE at pH 4.5 and pH 8.3 and a single peak on HPLC size exclusion and cation exchange columns. The purified lectin was specific for both T-antigen and N-acetyl-D-lactosamine, markers for various carcinomas, in addition to N-acetyl-D-galactosamine, asialofetuin and fetuin. This lectin reacted strongly with red blood cells (RBCs) from human ABO blood groups and rat. It also reacted with rabbit, sheep, goat and guinea pig RBCs. The lectin is a glycoprotein having no metal ion requirement for its activity. Denaturing agents such as urea, thiourea and guanidine-HCl had no effect on its activity when treated for 15 minutes. AVL showed significant antiproliferative activity towards HB98 and P388D1 murine cancer cell lines. It also exerted antifungal activity against phytopathogenic fungi Botrytis cincerea and Fusarium oxysporum but not against Rhizoctonia solani, Trichoderma reesei, Alternaria solani and Fusarium graminearum.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

Affinity purification and characterization of a seed lectin from Crotalaria medicaginea

Purification of lectin from the seeds of Crotalaria medicaginea Lamk by affinity chromatography on asialofetuin-linked amino activated silica, yielded a single band on non-denatured PAGE at pH 4.5 and 8.3 and, a single peak on HPLC size exclusion and cation exchange columns. The molecular mass of the native C. medicaginea lectin was determined to be 125 kDa by gel filtration. In SDS-PAGE, the lectin migrated as a single band of M(r) 31.6 kDa under reducing and nonreducing conditions, indicating that it is a tetramer of apparently identical subunits. It agglutinated red blood cells (RBCs) from rabbit and human ABO blood groups. It also reacted with RBCs from rat, sheep, goat and guinea pig but after desialylation with neuraminidase. The hemagglutination activity of the lectin was inhibited by D-galactose and its derivatives. Amino acid analysis showed that lectin was rich in aspartic and glutamic acid and, did not contain sulphur containing amino acids. The lectin is a glycoprotein having 1.41% of neutral sugars. It is labile at temperature above 60 degrees C. It needs divalent cations for its activity, as a loss of activity was observed on removal of Ca2+ and Mn2+. Denaturing agents like urea, thiourea and guanidine-HCl have no effect on its activity.     

Isolation,purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.     

First report of an arabinose-specific fungal lectin

To date, arabinose-binding lectins have been reported only from the human opportunistic pathogen Pseudomonas aeruginosa, the plant aggressive pathogen Ralstonia solanacearum, and the sponge Pellina semitubulosa. An arabinose-binding lectin with mitogenic activity toward splenocytes and a high specific hemagglutinating activity was isolated in the present study from a wild discomycete mushroom, Peziza sylvestris. The maximal mitogenic activity was induced by a lectin concentration of 8 microM. The lectin was a single-chained protein with a molecular mass of 20 kDa. Its N-terminal sequence showed only slight resemblance to other mushroom lectins. It was adsorbed on both diethylaminoethyl-cellulose and carboxymethyl-cellulose. Unlike previously reported mushroom lectins, the hemagglutinating activity of the lectin was inhibited by arabinose, but not by a large variety of other carbohydrates. The lectin activity was adversely affected in the presence of 0.05 M NaOH or 0.025 M HCl, and when the ambient temperature was elevated above 35 degrees C.     

A novel lectin from the wild mushroom Polyporus adusta

A lectin with antiproliferative activity toward tumor cell lines and mitogenic activity toward splenocytes was isolated from the mushroom Polyporus adusta. The lectin was composed of two identical subunits each with a molecular weight of 12 kDa. It was adsorbed on both DEAE-cellulose and Q-Sepharose and unadsorbed on CM-Sepharose. The hemagglutinating activity of the lectin was inhibited by turanose and by a large variety of other carbohydrates. It was adversely affected in the presence of NaOH or HCl at a concentration of 7.5mM and above, and when the ambient temperature was raised above 70 degrees C. All divalent and trivalent metallic chlorides tested at 1.25-10mM including CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3), did not alter the hemagglutinating activity of the lectin. FeCl(3) at 10mM caused the hemagglutinating activity to increase by 100%, but it did not change the lectin activity when tested at lower concentrations up to 5mM.     

A mushroom lectin from ascomycete Cordyceps militaris

A mushroom lectin has been purified from ascomycete Cordyceps militaris, which is one of the most popular mushrooms in eastern Asia used as a nutraceutical and in traditional Chinese medicine. This lectin, designated CML, exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes. SDS-PAGE of CML revealed a single band with a molecular mass of 31.0 kDa under both nonreducing and reducing conditions that was stained by silver nitrate, and a 31.4 kDa peak in a Superdex-200 HR gel-filtration column. The hemagglutination activity was inhibited by sialoglycoproteins, but not in by mono- or disaccharides, asialoglycoproteins, or de-O-acetylated glycoprotein. The activity was maximal at pH 6.0–9.1 and at temperatures below 50 °C. Circular dichroism spectrum analysis revealed that CML comprises 27% α-helix, 12% β-sheets, 29% β-turns, and 32% random coils. Its binding specificity and secondary structure are similar to those of a fungal lectin from Arthrobotrys oligospora. However, the N-terminal amino acid sequence of CML differs greatly from those of other lectins. CML exhibits mitogenic activity against mouse splenocytes.     

Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants

Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.     

Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion

The mannose-specific snowdrop lectin (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph. The ability of GNA to act as a carrier protein to deliver an insecticidal spider venom neurotoxin (Segestria florentina toxin 1: SFI1) to the haemolymph of lepidopteran larvae was investigated. Constructs encoding SFI1 and an SFI1/GNA fusion protein were expressed in Pichia pastoris. The insecticidal activity of purified recombinant proteins on injection was found to be comparable to published values for SfI1 purified from spider venom [Toxicon 40 (2002) 125]. Whereas neither GNA nor SFI1 alone showed acute toxicity when fed to larvae of tomato moth (Lacanobia oleracea), feeding SFI1/GNA fusion at 2.5% of dietary proteins was insecticidal to first stadium larvae, causing 100% mortality after 6 days. The protein also showed a significant, dose dependent, toxicity towards fourth and fifth stadium larvae, with growth reduced by up to approximately 90% over a 4-day assay period compared to controls. Delivery of intact SFI1/GNA to the haemolymph in these insects was shown by western blotting; haemolymph samples from fusion-fed larvae contained a GNA-immunoreactive protein of the same molecular weight as the SFI1/GNA fusion. SFI1/GNA and similar fusion proteins offer a novel and effective approach for delivering haemolymph active toxins by oral administration, which could be used in crop protection by expression in transgenic plants.     

Purification and characterization of tyrosinase from gill tissue of Portabella mushrooms

A group of tyrosinase isoforms with isoelectric points between 4.9 and 5.2 was isolated from gill tissue of Portabella mushrooms. Use of protease inhibitors was not able to increase the amount of latent forms significantly in crude extracts or to preserve latent tyrosinase activity during purification. The tyrosinase in gill tissue extracts showed latent activity above pH 5.5 and suppressed or displayed no latent activity below pH 5.5 when assayed in the presence of SDS. The purified isoforms showed monophenolase activity toward 4-hydroxyanisole but practically no activity toward tyrosine or tyramine. The purified isoforms showed greater activity toward catechol than either 4-methylcatechol, dopa, dopamine, chlorogenic acid, t-butylcatechol, or catechin. The Km for catechol was similar for the group of isolated isoforms (4.3 mM) compared to the isoforms in crude extracts (5.3 mM). Crude extracts showed several isoforms ranging from 50 to 230 kDa after partially denaturing SDS PAGE, while the purified isoforms showed molecular weights of 70 kDa.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.