Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed

A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca²⁺ and 0.4 mol Mn²⁺ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.     

从绿藻礁膜（Monostroma nitidum Wittr）分离半乳糖专一的凝集素

礁膜（Monostroma nitidum Wittr）经 25%～80%硫酸铵分级、DEAE-纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到纯化礁膜凝集素（Monostroma nitidum lectin,MNL）,在SDS-PAGE上显示单一蛋白染色带. 用Sephadex G-200层析测得其分子质量为66.6 kD, 用SDS-PAGE测得其分子质量为66.2 kD．该凝集素可以凝集人A、B、AB、O型红细胞,且凝集活性相同. 在对人（A、B、AB、O）、兔、鲤、鲫、鼠、羊、鸡、狗的红细胞凝集作用中,兔凝集作用最强．该凝集素在pH 4.00～10.53范围内均有活性,但在pH 5.20～9.40范围内活性最大．经100 ℃热处理30 min后,该凝集素对兔红细胞血凝活性保留25%,活性最大的温度范围为25～55 ℃．MNL被EDTA抑制,最小抑制浓度为3.13 mmol/L,但对 Ca^２＋和Mg^２＋不敏感．该凝集素凝集兔红细胞的作用不被D -果糖、D-甘露糖、D-葡萄糖、蔗糖、麦芽糖、γ-球蛋白、牛甲状腺球蛋白所抑制,但被D- 半乳糖和乳糖抑制,最小抑制浓度分别为5 mmol/L和2.5 mmol/L．     

Purification and Characterization of Lectin from Lathyrus sativus

Lectin has been isolated and purified from Lathyrus sativus using ammonium sulphate precipitation followed by affinity chromatography. The molecular weight as determined by HPLC was found to be 42kD. The lectin is a tetramer, consisting of two types of subunits of which the heavier subunit consists of 2 polypeptides of mol wt of about 21 kD and 16 kD while the smaller subunits consists of two polypeptides of about 5kD as revealed by SDS-PAGE. The most potent sugar inhibitor of the Lathyrus lectin was found to be α-methyl D-mannoside. The N-terminal amino acid sequence was similar to that of pea lectin sequence.     

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

The lectin from Euonymus europeus seeds was purified by adsorption onto insoluble polyleucyl hog A + H blood group substance and subsequent elution with lactose. The isolated lectin formed three lines in immunoelectrophoresis against rabbit antisera to the crude seed extract and showed three components on electrophoresis in acrylamide gel at pH 9.4. In analytical isoelectric focusing the purified lectin had six closely spaced bands with pI from 4.3 to 4.7. It sedimented as two peaks: a big symmetrical peak with s_20,w⁰ of 7.8 and another small, diffuse moving peak. The intrinsic viscosity was 0.057 dl/g and the M_r calculated from the sedimentation coefficients, intrinsic viscosity, and V? of 0.71 was about 166,000. In sodium dodecyl sulfate, it gives subunits of M_r 17,000 and 35,000; 20% of the 35,000 subunit resists reduction by dithiothreitol in 7 m guanidine-HCl. The Euonymus lectin is a glycoprotein containing 4.8% d-galactose, 2.9% d-glucose, and 2.8% N-acetyl-d-glucosamine. The purified lectin precipitated well with B and H blood group substances and with the P1 fraction of blood group B substance but not with A₁ substances. It precipitated poorly with Le^a and Le^b and precursor I blood group substances. Inhibition of precipitation with milk and blood group oligosaccharides showed the lectin to be most specific for blood group B oligosaccharides having the structure: dGalα1 → 3[lFucα1 → 2]dGalβ1 → 3 or 4dGlcNAcβ→. It is also inhibited by blood group H oligosaccharides but to a lesser degree. For 50% inhibition of precipitation, 3.5, 850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively are required. The B and H specificities are an intrinsic property of a single lectin site since absorption and elution from an H immunoadsorbent gave material with B as well as H specificity. Millipore-filtered crude extracts of Euonymus europeus preserved with 0.02% sodium azide are stable in the refrigerator for many months and can be used for quantitative precipitin and for quantitative inhibition assays, results being the same as with purified lectin.     

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied.MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed.ResultsThe purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (K_a=4.9 (±0.2)×10⁴ M^?1), which indicated static quenching (Stern-Volmer (SV) constant K_sv=4.6 (±0.2)×10⁴ M^?1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa.ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored.General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.     

Purification and characterization of lectin II from Ulex europaeus seeds and an immunochemical study of its combining site.

The lectin II from Ulex europaeus seeds was purified by adsorption on insoluble polyleucyl hog A + H blood group substance and elution with 35% ethylene glycol, and by chromatography on ?-aminocaproyl-fucosyl-amine-agarose. In immunodiffusion against rabbit antiserum to the crude extract, the isolated lectin formed one line which fused with one of the five formed by crude extract. The purified lectin showed two bands on acrylamide electrophoresis under alkaline or acid conditions but only one band of molecular weight 23,000 if the electrophoresis was in the presence of 0.1% sodium dodecyl sulfate at pH 8.8. The agglutinating and precipitating abilities are abolished by EDTA and can be restored by bivalent cations. The purified lectin precipitated to different extents with blood group A₁, A₂, B, HLe^b, Le^a, and I precursor substances and with acid- or Smith-degraded substances. Inhibition of precipitation indicated that the lectin site was unusual in that it interacted most strongly with the h-specific oligosaccharide

and with 2′-fucosyllactose, followed by β1 → 4 linked oligomers of dGlcNAc. Molecular models showed that all these inhibitors have a similarity in three-dimensional structures that could account for their activities.     

Haemagglutinin inTriticale

Saline extracts of several varieties ofTriticale had haemagglutinin activity against rabbit, rat and fowl erythrocytes. In contrast to the wheat germ lectin theTriticale lectin was inactive against human B, 0 blood group type erythrocytes and rather high concentrations of the lectin are needed to agglutinate human A blood group type erythrocytes. TheTriticale lectin was purified about 20-fold with a 10% recovery of activity from one of the varieties (DTS 138) by (NH₄)₂SO₄ fractionation followed sequentially by chromatography on DEAE-cellulose and sulphopropyl-Sephadex. Approximately 4 μg of the purified lectin caused visible agglutination with trypsinised rabbit erythrocytes. Among a variety of sugars tested D-glucose, D-mannose and N-acetyl-D-glucosamine (2·5-7·5mM) caused inhibition of agglutination.     

Isolation and characterization of a lectin from the seeds of Dioclea grandiflora (Mart.)

By a combination of solubility fractionation, affinity and molecular-sieve chromatography, a lectin preparation containing several closely related lectin components of different isoelectric point was isolated from the seeds of Dioclea grandiflora Mart. The lectins showed a carbohydrate specificty for D-mannose (D-glucose)-binding and had a requirement for the presence of Ca²⁺ and Mn²⁺. The results of preliminary characterization studies showed that the D. grandiflora lectins had similar properties to those of concanavalin A, the lectin from the seeds of Canavalia ensiformis, a plant also belonging to the tribe Diocleae. Thus the D. grandiflora lectins contained no covalently bound carbohydrate and had an amino-acid composition characterized by a low content of methionine and the virtual absence of cysteine. Above pH 4.8 they had molecular weight of about 100,000, while below pH 3.1 they were dissociated to half-molecules. Between these two pH values there was a fast association-dissociation equilibrium for the two species. In dissociating solvents, three subunits were obtained of the approximate size of 25–26,000, 13–14,000 and 8–9,000. The lectins from C. grandiflora similar to concanavalin A were more distantly related to the lectins obtained from the members of the tribe Vicieae although these were also specific for D-mannose (D-glucose)-binding.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

Tryptophan207 is involved in the GTP-dependent conformational switch in the α subunit of the G protein transducin: Chymotryptic digestion patterns of the GTPγS and GDP-bound forms

The limited proteolytic pattern of transducin,G _t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the α _t subunit were identified. The α _t subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas α _t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγ _t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γ _t subunit was not cleaved and only a small portion of β _t was digested into several fragments. In order to determine which proteolytic fragment of α _t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG _t previously³²P-labeled at Cys³⁴⁷ by petrussis toxin-catalyzed ADP-ribosylation. The³²P-label was mainly associated with the α _t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not³²P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of α _t , at Leu¹⁵ and Leu¹⁹. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of α _t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp²⁰⁷ in a conformation-dependent manner. Trp²⁰⁷ of α _t -GTPγS was resistant to proteolysis but α _t -GDP and the 38 kD fragments of α _t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp²⁰⁷ changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.     

Isolation and characterization of a group of isolectins with galactose/N-acetylgalactosamine specificity from hemolymph of the giant silk moth Hyalophora cecropia

A series of isolectins from Hyalophora cecropia was purified by affinity chromatography on d-GalNAc-Sepharose. The yield of the lectin was 0.4 mg/ml of hemolymph and the concentration was about the same in larvae and pupae. The molecular weight of the lectin was around 160,000 and the sizes of the subunits were 41 and 38 kD for the A and the B chains, respectively. The isolectins are believed to be tetramers with varying proportions between the two subunits. Inhibition studies indicate that the A chain has a binding site with specificity for d-Gal/d-GalNAc while the B chain has specificity for an unknown structure on rabbit erythrocytes not necessarily of carbohydrate nature. A d-Gal/d-GalNAc specific lectin was found also in Anthereae pernyi but the yield was only 0.03 mg/ml of hemolymph.     

Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Purification,characterization of mosquito larvicidal lectin from Annona muricata and its eco-toxic effect on non-target organism

The present study investigates the purification and characterization of mosquito larvicidal lectin from the seed kernel extract of Annona muricata and toxic effects on non-target organism Chironomus costatus. Soursop lectin was purified by anion-exchange column chromatography using DEAE-cellulose with approximately molecular mass of 260 kDa and with seven distinct subunits (16, 18, 21, 22, 24, 73 and 95 kDa). Soursop lectin highest Hemagglutination (HA) titer value of 128 was recorded against hen indicator RBC type with the influence of different divalent cations such as Ca²⁺, Ba²⁺ and Mn²⁺. The lectin mediated HA activity was highly inhibited by monosaccharides of glucose and mannose and disaccharides such as trehalose and melibiose. It was found to be EDTA sensitive (at 30 mM), pH-dependent (between 6–9) and heat-labile (upto 60 °C). A significant homology for soursop lectin was recorded to leguminous seed lectin from Psophocarpus scandens in MALDI-TOF-MS analysis with 66 % of sequence coverage. Finally, the toxicity bioassay of soursop lectin resulted in 100 % larval mortality for A. aegypti at 48 h whereas only 10 % mortality recorded to non-target organism C. costatus. Therefore, we concluded that soursop lectin could be a potent insecticidal agent in integrated pest management for controlling various insect pests.     

An acid-tolerant lectin coupled with high Hg2+ potentiated hemagglutination enhancing property purified from Amanita hemibapha var. ochracea

A 37.4 kDa acid tolerant lectin was isolated and purified from dried fruiting bodies of Amanita hemibapha var. ochracea designated as AHL. The lectin was not adsorbed on DEAE-cellulose, but rather adsorbed on S-Sepharose and subjected to gel filtration by fast protein liquid chromatography on Superdex 75. The purified lectin was immune from inhibition activities of metal ions. More over, AHL exhibited high agglutination activity on rabbit erythrocytes with accelerating Hg²⁺ ions concentration. Partial peptide sequence analysis (VSNNLLTGPKVVR) of this lectin showed relative similarity to phosphoenolpyruvate carboxykinase [ATP]-like protein as predicted from Fragaria vesca subsp. Vesca. Interestingly, AHL displayed a strong affinity toward α-Lactose, making our study the first report associating Amanita species’ lectin specificity for α-Lactose to the best of our knowledge.     

A phytohaemagglutinin from the Azolla-anabaena symbiosis

Proteins recovered from cell-free extracts of the Azolla-Anabaena azollae symbiosis exhibited haemagglutination activity; galactose was the most effective carbohydrate tested in preventing haemagglutination. Extracts of cyanobacteria-free Azolla also caused haemagglutination but extracts of free-living or symbiotic Anabaena azollae did not. Symbiotic Azolla plants grown on NO₃^? showed lower haemagglutination activity than did those grown on N₂; activity increased on removal of NO₃^?. The lower activity of the NO₃^?-grown material may be due to NO₃su? exerting a direct effect on lectin activity/synthesis, or it may act indirectly by inhibiting the development of Anabaena which in turn affects the Azolla lectin. The purified lectin was shown to be composed of 6 sub-units, each of M.W. 21000.     

A new Bauhinia monandra galactose-specific lectin purified in milligram quantities from secondary roots with antifungal and termiticidal activities

This work describes the purification in milligram quantities of a lectin from Bauhinia monandra secondary roots (BmoRoL) and its antifungal and termiticidal activities. The BmoRoL (6.2 mg) was isolated through ammonium sulfate fractionation and affinity chromatography on guar gel. Native lectin was resolved as a single band on polyacrylamide gel electrophoresis for basic proteins. Under denaturing and reducing conditions it appeared as a unique glycosylated polypeptide of 26 kDa. The highest agglutination activity of BmoRoL was found with glutaraldehyde-treated rabbit erythrocytes. BmoRoL showed antifungal activity against phytopathogenic species of Fusarium and was more active on Fusarium solani. The lectin also showed termiticidal activity on Nasutitermes corniger workers and soldiers with LC₅₀ of 0.09 and 0.395 mg ml⁻¹ for 12 days. In conclusion, BmoRoL is a new antifungal and termiticidal lectin that can be purified in milligram quantities and has potential biotechnological application for control of agricultural pests.     

Purification and Characterization of Phosphoribulokinase from Immature Pods of Brassica

Phosphoribulokinase (ATP:D — ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19; PRuK) from immature pods of Brassica was purified to apparent homogeneity with about 31% recovery using ammonium sulphate fractionation, gel filtration through Sepharose CL-6B and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme, having molecular mass of about 180 kD, was heterotetramer with subunit molecular mass of 48, 47, 41 and 33 kD. The enzyme had an absolute requirement for a divalent cation Mg²⁺ and a monovalent cation K⁺for optimal activity. At optimum pH of 8.0–8.4, the enzyme showed typical hyperbolic response for both the substrates with Km values of 333 μM and 100 μM, respectively for Ru5P and ATP. The enzyme was inhibited by RU-1, 5-P₂, 6-phosphogluconate and AMP, and activatded by glu-1-P, glu-6-P and Pl. RU-1, 5-P₂ and 6-phosphogluconate inhibited the enzyme competitively with respect to Ru5P and non-competitively with respect to ATP. It appears that the activity of the Brassica pod enzyme besides being controlled at the level of metabolites, is regulated by light and energy status of the cell.     

Soybean lectin and related proteins in seeds and roots of le and le soybean varieties

The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le⁺) and lectin negative (Le⁻) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le⁺ varieties. There is no detectable lectin in the protein bodies of Le⁻ cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le⁺ and Le⁻ plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4-day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves.