Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.     

Assay of lectin binding sites in colon cancer-associated mucins: comparison of fluorescence microscopy with a quantitative chromatographic technique

We have developed a technique for quantitation of binding of fluorescent lectins to glycoconjugates in specimens of tumors derived from cultured human colorectal cancer cells. Tumor cells were injected subcutaneously into nude mice, giving rise to xenografts that resemble primary human colorectal cancers. The tumors were extracted with saline and were subjected to dialysis and lyophilization. Standardized amounts of the tumor extract were then incubated with fluorescent lectins and subjected to gel permeation liquid chromatography to separate lectin bound to high molecular weight glycoproteins from free (unbound) lectin, and were quantitated using a spectrofluorometer. This assay permitted quantitative measurement of the lectin bound to high molecular weight glycoconjugates such as mucin. The results of this assay were compared with the standard histochemical assessment of tissue labeling by fluorescent lectins. A close correlation between the two techniques was found, especially when little or no labeling was present. Greater variations were observed at higher levels of labeling. The quantitative assay confirms that lectins bind to high molecular weight mucin-type glycoconjugates on fixed sections of tumors, and supports the use of semi-quantitative histochemical assessments of tissue labeling.     

Biochemical characterization and biosynthesis of the uterine milk proteins of the pregnant sheep uterus

The uterine milk proteins (UTM-proteins), a pair of basic glycoproteins with similar isoelectric points and molecular weights (57,000 and 55,000), are secreted by the endometrium of the pregnant ewe. Peptide mapping of the two species of UTM-proteins demonstrated them to be structurally related. Furthermore, pulse-chase and continuous-labeling experiments indicated that both are produced from a common precursor of lower molecular weight. Purified UTM-proteins were found to be rich in basic amino acids, low in tyrosine, and apparently lacking in tryptophan. The proteins were about 5.6-5.7% carbohydrate by weight and bound the lectin, concanavalin A. UTM-proteins synthesized in vitro incorporated D-[3H]glucosamine. Analysis of [3H]glucosamine-labeled glycopeptides of Pronase-digested UTM-proteins by gel filtration indicated that most radioactivity is associated with one size class of oligosaccharide. UTM-proteins secreted by the endometrium in the presence of tunicamycin, an N-glycosylation inhibitor, were of lower molecular weight than those from control endometria, indicating that sugar chains are attached to the protein core via N-linkages to asparagine. UTM-proteins synthesized in culture incorporated [32P]orthophosphate, and tunicamycin inhibited this incorporation. Analysis of hydrolyzed UTM-proteins by paper chromatography indicated that much of the 32P was associated with mannose 6-phosphate. Because this moiety is the so-called lysosomal recognition marker and is present on uteroferrin, the acid phosphatase of porcine uterine secretions, we tested UTM-proteins for several enzymatic activities characteristic of lysosomes, but none was found. In conclusion, the UTM-proteins are related glycoproteins that, like porcine uteroferrin, contain mannose 6-phosphate, a result which suggests that secretion of glycoproteins with phosphorylated oligosaccharide chains may be a common feature of the progestational uterus.     

IgA1 is the premier serum glycoprotein recognized by human galectin-1 since T antigen (Galbeta1-->3GalNAc-) is far superior to non-repeating N-acetyl lactosamine as ligand

Human heart galectin-1 (HHL) was separated by high pressure liquid chromatography from endogenous glycoproteins co-purified with it during affinity chromatography. These glycoproteins offered excellent ligands for HHL binding and were rich in T antigen (Galβ1 → 3 GalNAc-) of O-linked oligosaccharides. In enzyme linked lectin assay and hemagglutination inhibition assay, human IgA1, bovine fetuin and other O-glycosylated T antigen-bearing glycoproteins bound to the lectin efficiently in contrast to single N-acetyl lactosamine (LacNAc)-bearing N-linked oligosaccharides released from them and to IgG which is not O-glycosylated. HHL binding to IgA1 and fetuin was unaffected by removal of their N-linked oligosaccharides by -mannosidase. When immobilized, O-glycosylated serum proteins but not IgG could capture HHL from its solutions. Desialylated or polymeric IgA1 was better inhibitor than monomeric IgA1. The findings suggest a possible role for galectin-1 in anchoring of microbial and cancer cells known to be rich in T antigen, in high serum IgA1 turn over and in tissue sequestering of IgA1 immune complexes especially after their microbial desialylation in IgA nephropathy and other immune complex-mediated disorders.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

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.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Mitochondrial biogenesis: do liver mitochondria contain glycoproteins and glycosyltransferases?

1. Subcellular fractions isolated from livers of 19-day-old chicken embryos were analyzed in order to assess whether liver mitochondria contained glycosylated proteins or had mannosyl- or sialyl-transferases that could transfer sugars to mitochondrial macromolecules. 2. Proteins in liver mitochondrial membranes and matrix fractions were screened for their affinities for concanavalin A (Con A). 3. After separation by gel electrophoresis under denaturing conditions, a significant number of the proteins bound [125I]Con A, and the binding of the lectin was substantially inhibited by alpha-methyl-D-mannoside. 4. In addition, radio-iodinated matrix proteins were screened for lectin-binding properties by chromatography on Con A covalently linked to agarose. 5. A number of proteins, representing 14% of those loaded onto the column, became tightly bound to the agarose-linked lectin, and the molecular weights of several of those proteins are reported. 6. Mannosyltransferase activities were measured in fractions highly enriched for mitochondria. 7. In the reactions, mannose was transferred from guanosine diphosphomannose to materials insoluble in 0.3% trichloroacetic acid or in chloroform:methanol (2:1). 8. The fractions also catalyzed the transfer of mannose to materials extractable in chloroform:methanol and which migrated with the Rf of dolichol phosphate on Silica Gel H. 9. Dolichol phosphate stimulated the transfer of mannose to those materials extractable in the organic solvents. 10. Marker enzyme analyses indicated that the mannosyl transferase activity in the mitochondrial fraction could not be accounted for entirely by contaminating microsomal membranes. 11. Although sialyltransferase activity was detected also in the mitochondrial fractions, the levels of the activity and the kinetics of the reactions indicated that Golgi membranes were most likely the sources of the enzyme.     

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.     

Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones.

The gallbladder bile of patients with cholesterol gallstones contains pronucleating proteins which accelerate precipitation of cholesterol crystals from bile. In this study we have improved the purification procedure developed earlier for these nucleating proteins and have now identified the nature of these proteins. Gallbladder bile from patients with cholesterol gallstones was applied to concanavalin A affinity columns. The ConA-binding glycoprotein fractions containing the nucleating proteins were then separated by FPLC (fast protein liquid chromatography) using a Superose 12 gel filtration column. Nucleating activity was detected in the high molecular weight (FPLC-1) as well as in the low molecular weight fractions (FPLC-3). Investigation of the high molecular weight fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution and amino acid sequencing suggested that these proteins were immunoglobulins. Immunostaining of Western blots with specific monoclonal antibodies identified the presence of immunoglobulin (Ig) M and IgA in the FPLC-1 fraction. These immunoglobulins were further purified by affinity chromatography employing an antibody exchanger (ABx) column which specifically binds immunoglobulins. There was no reduction in the cholesterol nucleating activity in the Abx-bound fraction compared to FPLC-1. Additional studies showed that the FPLC-1 fraction was significantly more potent than the ConA glycoproteins from either rapid and slow nucleating biles. Also the number of crystals formed was significantly greater in the FPLC-1 fraction isolated from cholesterol gallstone biles than from the FPLC-1 fraction from control patient biles. Commercially obtained IgM and IgA had no effect on nucleation, but IgM isolated from the serum of patients with Waldenstrom's macroglobulinemia did accelerate the nucleation of cholesterol. We conclude that the IgM and possibly IgA are pronucleating proteins and may be important in the pathogenesis of cholesterol gallstones in man.     

Growth factor for cardiac hypertrophy

Cardiac size can be regulated by the balance in activity between cardiac growth factors and inhibiting factors, chalones. This study was undertaken to verify the role of the cardiac growth factor and its purification from hypertrophied hearts. For this propose the hypertrophied hearts of renovascular hypertensive rats were used. The purification was made by using an isoelectric focusing chromatography and the HPLC method. We examined the cardiac growth effect of the isolated fractions with cultured chicken embryonic cardiac myocytes. Simultaneously, the influence of these fractions on the cardiac cell cycle was examined by DNA analysis with the flow cytometric method. If the hearts were overloaded due to hypertension, the growth of the cardiac size could be induced by increased the level of five proteins with different molecular weight and with an isoelectric point of 8.3. The significant growth activities were observed at these five proteins compared to the absence of the fractions. For the appearance of these growth effect, it is necessary that the structure of the protein is there fundamentally as a form with a molecular weight of 27 k dalton. After addition of these isolated fractions, BrdU content is S and G2 phases by flow cytometry was increased. This change indicates that the cardiac myocytes are stimulated in form DNA synthesis.     

Microtubule associated proteins of goat brain

The microtubule associated proteins of goat brain were separated from tubulin on the basis of their thermostability and then fractionated by chromatography on Sepharose 4B column. Analysis of the fractions by SDS-Polyacrylamide gel electrophoresis and assay of their tubulin-assembly-promoting activity indicate that this activity resides primarily in the tauproteins (mol. wt. 55,000–70,000) and a class of even lower molecular weight (25,000–35,000) proteins. Electrophoresis of the microtubule associated protein fractions separated from tubulin by phosphocellulose chromatography are in agreement with the results obtained from fractionation on Sepharose 4B columns.     

THE AFFINITY OF THE FUCOSE-BINDING LECTIN FROM LOTUS TETRAGONOLOBUS FOR GLYCOPEPTIDES AND OLIGOSACCHARIDES ACCUMULATING IN FUCOSIDOSIS

Abstract— The affinity of the fucose-binding lectin from Lotus tetragonolobus for fuco-oligosaccharides accumulating in the brain and other tissues of a patient with fucosidosis was studied by two methods: by inhibition of the co-precipitation of the lectin with porcine stomach mucin and by one-step affinity chromatography on a column of the lectin bound to Sepharose-4B. Both methods indicated that the lectin had greater affinity for the disaccharide Fuc(α, 1-6)GlcNAc than for either the main fucosidosis storage material in brain, a fuco-dekasaccharide, or the heterogeneous fuco-glycopeptide fractions obtained from normal human and rat brain glycoproteins. Our results suggest that the fucose residue linked α(1-6) to the N -acetylglucosamine residue involved in the N -glycosidic linkage to asparagine is not available to the lectin in the intact N -glycosidic chains of normal brain glycopeptide fractions and that the lectin has poor affinity for the Fuc(α, 1-3)Glc N Ac linkage in rat brain glycoproteins.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Exploring the oviductal fluid proteome by a lectin‐based affinity approach

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel‐based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography‐tandem mass spectrometry (nLC‐ESI‐MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin‐bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin‐bound fraction were known glycoproteins (p‐value [FDR] = 1.45E‐31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin‐based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Purification and analysis of DNases of Tritrichomonas foetus: evidence that these enzymes are glycoproteins

Tritrichomonas foetus is the causative agent of trichomoniasis. In cattle, infection results in economic losses to the beef and dairy industries due to abortion and infertility. Soluble DNases of T. foetus that play a role in pathogenesis and are potential therapeutic targets, were extracted and purified utilising lectin affinity chromatography. The DNases were bound to and eluted from Concanavalin A (Con A)-sepharose indicating that they are glycoproteins with -linked mannose or glucose residues. The nature of the glycans carried on the eluted proteins in the fraction containing DNase activity was assessed using an enzyme-linked lectin assay. The lectin binding studies predict the presence of both N- and O-type glycans. Manganese was a potent (33%) activator of the DNase(s) whereas zinc inhibited enzyme activity by approximately 66%. The DNase(s) had a pH optimum of 4 and a molecular weight of 160 kDa. The DNase(s) were able to completely degrade DNA from animal, plant, fungal, yeast and bacterial sources, but did not significantly degrade RNA.     

棕尾别麻蝇(Sarcophaga peregrina)幼虫与蛹血淋巴凝集素的纯化与特性

用亲和层析法纯化了棕尾别麻蝇幼虫和蛹血淋巴凝集素。以兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗体、球球蛋白和甲状腺蛋白等三种亲和层析吸附剂纯化得到的幼虫凝集素是相同的,其分子量73kD左右。用甲状腺球蛋白为亲和配基纯化的蛹血淋巴凝集素由二种亚基组成,其分子量分别为30和32kD。幼虫和蛹血淋巴凝集素活性的抑制糖明显不同：乳糖、岩藻糖和N－乙酰半乳糖胺对幼虫血淋巴凝集素活性有抑制作用;而甘露糖胺、半乳糖胺和葡萄糖胺则对蛹血淋巴集素有一定抑制。而且,用兔红细胞吸附幼虫血淋巴凝集素为抗原制备的抗血清对蛹的凝集素活性无交叉反应,表明这两种凝集素是不相同的。虽然本文所纯化的麻蝇蛹血淋巴凝集素的分子量和Komano等报道的麻蝇蛹以及幼虫体壁 伤害诱导的凝集素SPL相同,但其糖的抑制特性有明显差异。     

Lectin affinity as an approach to the proteomic analysis of membrane glycoproteins

The aim was to determine the proportion of membrane glycoproteins captured using concanavalin A or wheat germ agglutinin lectin affinity chromatography. Digests of the isolated proteins were separated by reversed-phase liquid chromatography and analyzed by matrix-assisted laser desorption tandem mass spectrometry. The two lectins identified different groups of proteins with a broad range of molecular mass and p/ values, including a number of proteins that overlapped the two groups. Approximately 30% of the proteins were positively identified as containing domains that were predicted using standard bioinformatics methods to be characteristic of integral membrane proteins. This approach represents an effective method of surveying the membrane protein pool of mammalian cells for subsequent proteomic analysis.     

Polymorphism and glycoprotein character of Ricinus communis lectins purified by affinity chromatography]

Two lectin fractions (S20W = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM celluloes. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.