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Lectin-like activity from Persea americana
Abstract
An extract from the seeds of Persea americana possessed an erythro-agglutinating activity. The agglutinin was devoid of specificity for carbohydrates, but interacted readily with basic proteins or basic polyamino acids. The interaction between the agglutinin and egg-white lysozyme was not inhibited by chaotropic salts, but was sensitive to relatively low concentrations of urea. An affinity chromatographic procedure was developed in an effort to purify the agglutinin. Products from the chromatographic procedure were found not to contain higher specific agglutinating activities than the crude extract. Amino acid acid analyses of the extract showed the presence of relatively high proportions of glutamic and aspartic acids. In addition, the extract contained phosphorus and a visible chromophore. The agglutinin was resistant to detergents and denaturants, and proteases, nucleases, and other enzymes. The results suggest that, as opposed to other plant agglutinins, the active component from Persea is not a protein. Similarly, in contrast to many lectins, the agglutinin from Persea was not mitogenic for mouse lymphocytes. The agglutinin partially inhibited the mitogenesis of lymphocytes when the cells were treated with concanavalin A, or with bacterial lipopolysaccharide.
... (LCA) and Persea americana (PAA) were prepared and standardized as described elsewhere [7]. The lectins of Canat'alia ensiformis (conA), Helix pomatia (HPA), Limulus polyphemus (LPA), Solanum tuberosum (STA), Triticum l'ulgaris (TVA) and Ulex europaeus (UEA) were obtained from Sigma (Poole, U.K.). ...
... (LCA) and Persea americana (PAA) were prepared and standardized as described elsewhere[7]. The lectins of Canat'alia ensiformis (conA), Helix pomatia (HPA), Limulus polyphemus (LPA), Solanum tuberosum (STA), Triticum l'ulgaris (TVA) and Ulex europaeus (UEA) were obtained from Sigma (Poole, U.K.). ...
Phase I cells of Bordetella pertussis but not those of B. parapertussis, B. bronchiseptica or B. avium were agglutinated by Limulus polyphemus lectin. Most strains of B. pertussis but not those of the other species were also agglutinated by Helix pomatia lectin. In precipitation reactions between lectins and purified Bordetella lipopolysaccharide (LPS) preparations a similar pattern occurred. Lectin agglutination provides a rapid presumptive method for the differentiation of B. pertussis from B. parapertussis and other Bordetella species.
... Onde foi constatado que no pH 8,2 havia uma maior expressão da atividade hemaglutinante específica. Meade et al., (1980) com o extrato das sementes da espécie, onde foi cerificado a capacidade de realizar a aglutinação, outros testes foram realizados pelos autores onde foi confirmado que a substância presente no extrato não tinha especificidade por carboidratos e não se tratava de uma proteína. ...
... In P. americana extract we detected the presence of flavonoids, terpenes, tannins, and saccharides (Table 1), as reported by others (Antia et al., 2005). Although, the aqueous extract of this plant was shown to possess anti-inflammatory and analgesic activities (Adeyemi et al., 2002), lectins from its seeds were not mitogenic for mouse lymphocytes (Meade et al., 1980), and "persin" from avocado leaves was shown to have antifungal properties and to be toxic to silkworms (Oelrichs et al., 1995), immune enhancing properties of this plant has not been previously reported. In addition, we observed the presence of alkaloids, saponins, terpenes, tannins, and saccharides in P. virginica extracts; the presence of such compounds has been reported in Plantago species (Samuelsen, 2000). ...
Medicinal plants have been used for centuries and have become part of complementary medicine worldwide because of their potential health benefits. Some of their metabolites have been successfully used directly in the treatment and prevention of infectious diseases and cancer, or indirectly by stimulating the immune system. In the present study, we investigated the effects of methanol and aqueous extracts of the Mexican plants Ocinum basilicum, Persea americana, Plantago virginica, and Rosa spp. on in vitro rat lymphocyte proliferation. Methanol extracts of O. basilicum, P. americana, P. virginica, and Rosa spp. stimulated up to 80, 16, 69 and 66% lymphoproliferation, respectively, whereas their respective aqueous extracts induced up to 83, 48, 31 and 83% lymphoproliferation, as compared with untreated controls. The effect of O. basilicum aqueous extract at concentrations of 31.25, 62.5, 125 and 250 mu g/ml on lymphoproliferation was significantly different (p < 0.05) than the effects of P. americana and P. virginica at the same concentrations. We also observed that the lymphoproliferative effect of Rosa spp. aqueous extract was significantly higher (p < 0.05) than that of the methanol extract. Methanol and aqueous vehicles did not affect lymphocyte viability nor proliferation activity. The observed immunostimulatory effect may be of benefit in increasing the pool of lymphocytes in immunodeficiency patients.
... Polyphenolic compounds, such as tannic acid, are frequently found in extracts of higher plants and have broad antiviral spectra, inactivating a variety of virions extracellularly (Cheng and Yeung, 1988). Additionally, Animashaun et al. (1993) have shown that lectins from plants have the ability to prevent HIV-1 infection while Mead et al. (1980) have described a lectin-like activity from P. americana seeds. ...
Aqueous (PA1) and methanolic extracts (PA2a–d; PA3) from the tropical tree Persea americana Mill. (Lauraceae), were evaluated for their cellular toxicity and anti-HIV-1 activity both in virustatic and virucidal assays. With the exception of PA3 and PA2d, all extracts showed anti-HIV-1 activity at concentrations which were not toxic for the H9 indicator cells. From the methanol insoluble extract (PA2) four different fractions (PA2a–d) were obtained using reverse-phase column chromatography, and two of the fractions (b and c) showed detectable virucidal effect. One fraction (PA2a) showed virustatic effects inhibiting HIV syncytium formation and viral p24 antigen formation at concentrations which were not toxic for the indicator cells. The results demonstrate for the first time that extracts from P. americana leaves have moderate anti-HIV-1 activity in vitro.
Purpose
: Etiological diagnosis of pediatric patients with community‐acquired pneumonia is difficult. For therapy, one of the major problems is the difficulty in separating bacterial pneumonia which would benefit from antibiotics from non‐bacterial pneumonia. Therefore, we sought to identify potential biomarkers for distinguishing non‐bacterial pneumonia from bacterial pneumonia.
Experimental design
: Lectin microarray containing 91 lectins was used to screen serums from pediatric patients with pneumonia. Lectin‐based pull‐down assay combined with LC‐MS/MS was used to identify the potential biomarkers.
Results
: SNA‐I, a lectin binding preferentially to α2‐6 linked sialic acid residues, showed higher binding signals (near 42 kDa) in the mycoplasma pneumonia group, when compared with the other groups. A total of 18 proteins were identified with LC‐MS/MS. By Western blot analysis, we confirmed that the expression of haptoglobin‐related protein (HPR) was elevated in pediatric patients with pneumonia compared with normal children (P < 0.001). Furthermore, HPR was higher in the mycoplasma pneumonia group (P < 0.01) and the viral pneumonia group (P < 0.05), when compared with the bacterial pneumonia group.
Conclusions and clinical relevance
: Our results indicate that HPR is a potential serologic biomarker which can differentiate between bacterial pneumonia and non‐bacterial pneumonia. Detection of serum HPR might be useful for clinical diagnosis.
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The objective of this work was to analyse the behaviour of the positive catalase Campylobacter strains consequence of the interaction with the lectins. The results of agglutination tests with the lectins trialed, are indicators of the differences in the surface composition of the thermophilic Campylobacter strains, belonging to the C. jejuni, C. coli, C. hyointestinalis and C. lari species. Only 11,8% (29/245) of the strains were not typified due to the self-agglutination phenomenal. The remainder 88,2% (216/245) reacted to the eight lectins (Wisteria floribunda, Arachis hypogaea, Triticum vulgaris, Solanum tuberosum, Maclaura pomifera, Canavalis ensiformis, Bauhinia purpurea e Helix pomatia), allowing their grouping into 12 groups, each one with several sub-groups. The reaction was not specie exclusive and all the strains reacted positively to the Persea americana agglutinin. The utilization of the lectinogroups constitutes a good phenotypic identifier parameter in differenciating the glucid epitopes, becoming so a relevant epidemiological marker.
Ginkgo is native to Far East Asia – China, Japan and Korea. It is commonly planted in Buddhist and Taoist temples in East Asia. It has been introduced to other temperate areas in both hemispheres.
Extract of the seeds ofAnona reticulata, Camellia sinensis, Bauhinia acuminata, Cassia tomentosa, Malus sylvestris, Trigonella foenumgraecum, Cephalandra
indica, Lawsonia inermis, Anacardium occidentale, Mangifera indica, Nephelium litchi, Citrus lemoni, Aegle marmelos, Quassia
amara, Mimusops elengi, Achras sapota, Datura stramonium, Thevetia nerifolia, Persea americana andCycas circinalis, were screened for lectin activity by haemagglutination and haemagglutination inhibition assays. Lectin-like activity was
detected only in the seeds ofMangifera indica and Perseaamericana. The conventional methods for the isolation of lectins could not separate the haemagglutinins from the extracts. The properties
of these agglutinins suggest that they are not lectins.
Current literature suggests that lectins are becoming valuable reagents for the laboratory identification of infectious agents. The identification of bacteria, fungi, or protozoa may be confirmed if they bind to or agglutinate with certain lectins. Assay kits utilizing specific lectin agglutination reactions, coupled with conventional enzyme determinations, have been proposed for several bacteria. Factors such as specificity, stability, assay rapidity, and costs combine to make lectins attractive diagnostic reagents. It is likely that the use of lectins in diagnostic microbiology will continue to grow.
Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to type blood cells and are used in a wide spectrum of applications, including, in part, as carriers of chemotherapeutic agents, as mitogens, for fractionation of animal cells, and for investigations of cellular surfaces. Numerous studies have shown that lectins can be used to identify rapidly certain microorganisms isolated from a clinical specimen or directly in a clinical specimen. Lectins have been demonstrated to be important diagnostic reagents in the major realms of clinical microbiology. Thus, they have been applied in bacteriology, mycology, mycobacteriology, and virology for the identification and/or differentiation of various microorganisms. Lectins have been used successfully as epidemiologic as well as taxonomic markers of specific microorganisms. Lectins provide the clinical microbiologist with cost-effective and potential diagnostic reagents. This review describes the applications of lectins in clinical microbiology.
Agglutination tests with lectins indicated differences in the surface composition of strains of the thermophilic (optimum temperature 42 degrees C) Campylobacter species C. coli, C. faecalis, C. hyointestinalis, C. jejuni and C. laridis. All strains examined were agglutinated by the protein-reactive agglutinins of Mangifera indica (mango) and Persea americana (avocado) and a large proportion was also agglutinated by the carbohydrate-reactive lectins of Canavalia ensiformis (Jack bean) and Triticum vulgaris (wheat germ). Reactions with other lectins varied widely between strains, even of the same species and serotype. Lectin agglutination may be useful as a supplementary procedure for characterizing individual Campylobacter isolates for epidemiological purposes.
Current literature suggests that lectins are becoming valuable reagents for the laboratory identification of infectious agents. The identification of bacteria, fungi, or protozoa may be confirmed if they bind to or agglutinate with certain lectins. Assay kits utilizing specific lectin agglutination reactions, coupled with conventional enzyme determinations, have been proposed for several bacteria. Factors such as specificity, stability, assay rapidity, and costs combine to make lectins attractive diagnostic reagents. It is likely that the use of lectins in diagnostic microbiology will continue to grow.
Serogroups of Legionella pneumophila exhibited differential reactivities with plant agglutinin. Agglutination patterns were modified by growing the organisms in different media. The passage of four strains through guinea pigs did not result in altered reactivities with lectins or with plant agglutinins.
The screening of staphylococci with a panel of 14 lectins and extracts demonstrating lectin-like activity led to the development of a rapid agglutination slide test for the differentiation of certain coagulase-negative staphylococci and human strains of Staphylococcus aureus. The coagulase-negative staphylococci were agglutinated by agglutinins from Mangifera indica, Triticum vulgaris, and crude Limulus polyphemus. The test is rapid, requiring only 5 to 15 min to identify an unknown strain of staphylococci, as opposed to the 4 to 16 h required to perform the conventional tube coagulase test.
Oleic acid and dioleoyl phosphatidic acid at low concentrations (20 and 0.5 mu g/ml, respectively) agglutinate rabbit and rat erythrocytes, while dioleoyl phosphatidylcholine is not hemagglutinating up to 0.5 mg/ml. Palmitic acid is not a hemagglutinin and dipalmitoyl phosphatidic acid is a very poor one. A polar lipid fraction obtained from calf thymocytes and a commercial preparation of gangliosides also exhibit pronounced hemagglutinating activity. Modification of the erythrocytes by either trypsin or neuraminidase causes a marked increase in agglutination only with oleic acid, whereas glutaraldehyde fixation of the cells significantly decreases agglutination with oleic acid, dioleoyl phosphatidic acid and calf thymocyte lipids. None of the lipids tested agglutinate freshly drawn human and sheep erythrocytes, but agglutination occurs following fixation of the sheep cells with glutaraldehyde. Lipid-mediated hemagglutination is strongly inhibited by fetuin and bovine submaxillary mucin (0.5 mg/ml). Defatted bovine serum albumin, also at 0.5 mg/ml, inhibits agglutination by oleic acid, whereas agglutination by other lipids is only poorly inhibited if at all. Monosaccharides at concentration up to 0.25 M do not inhibit the hemagglutinating activity of the lipids. Comparison of the hemagglutinating properties of lipids and lectins raises the possibility that the agglutinating activity of crude biological extracts which is not inhibited by mono- or oligosaccharides may be due to lipid constituents. Since agglutination by lipids is species specific, they may serve as mediators in intercellular recognition. The mechanism of lipid-mediated hemagglutination is discussed in terms of current concepts of the fusogenic activity of these compounds.
Agglutination of 271 strains of Yersinia enterocolitica and related species grown at 37 degrees C by a 0.01% dilution of the agglutinin from Mangifera indica was correlated with the presence of the virulence plasmid. The study of YadA mutants suggested that the YadA protein is the target of the plant agglutinin.
A simple method is presented by which, with the diffusion of trypan blue into the nucleus as a criterion of cell injury, it is possible to study quantitatively the effect of various agencies upon the small thymus cells and upon the tissue lymphocytes. Preliminary studies with this method have led us to the following conclusions, which, however, unless otherwise stated, may be taken as applying only to the lymphocytes of the rat thymus. 1. The small thymus cells, when suspended in balanced phosphate solutions, show no distinct reaction to variations in hydrogen ion concentrations ranging between P(h) 7.0 and P(H) 7.8. Beyond P(H) 7.0 there is a sudden increase in the permeability of the cells to the dye; plasmolysis of the cells occurs when the alkalinity exceeds P(H) 8.0. 2. Heating to 49 degrees or 50 degrees C. is accompanied by a critical increase in the permeability of the cells to the dye. 3. The injury caused by lack of oxygen can be demonstrated by the increase in the number of stained cells. 4. The addition of serum to suspensions of thymus cells or tonsil lymphocytes greatly inhibits the diffusion of the trypan into the cells. The protection afforded is roughly proportionate to the amount of serum added. Gelatin also exerts a marked protective influence; egg albumin affords a partial protection; starch and gum arabic are inert. Hemoglobin and cholesterol do not modify the stainability of the cells. Arsenious sulfide in weak concentrations partially inhibits the diffusion of the dye. Colloidal iron is without effect, and is precipitated about the cells. 5. The toxicity of the photodynamic substance, hematoporphyrin, and of an impure chlorophyll solution in the presence of sunlight could be strikingly demonstrated by the greatly increased permeability of the cells to the stain. 6. Acute and chronic inanition produces an increased fragility of the cells. The protective power of the serum in acute starvation appears to be increased. 7. The small thymus cells of old animals are more readily injured than are those of young ones, as indicated by the increased proportion of stained cells. 8. The method has been applied to the demonstration of the action of cytotoxic immune sera for rat thymus cells and for human tonsil lymphocytes in vitro. Further experiments dealing with the question of specificity are in progress. The cytotoxins are inactivated by the addition of complement. Thermostabile cytagglutinins have also been produced.
A receptor for prolactin and other lactogenic hormones (human growth hormone and placental lactogens) has been solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction isolated from pregnant rabbit mammary glands. The receptor remained in solution after centrifugation at 200,000 x g for 2 hours. Triton X-100 at concentrations higher than 0.01% (v/v) affected the physical properties of ¹²⁵I-prolactin, whereas ¹²⁵I-human growth hormone (hGH) was unaffected and, therefore, the latter has been used in studies of binding to the prolactin receptor in situations where ¹²⁵I-prolactin could not be used. It was possible to use ¹²⁵I-hGH for binding studies because this hormone has the same lactogenic potency as prolactin does in the rabbit and the prolactin receptor cannot discriminate between these two hormones. When an aliquot of soluble receptor protein was incubated with ¹²⁵I-hGH, the hormone-receptor complex was detected in the void volume of Sephadex G-100 column or, more rapidly, by precipitation with polyethylene glycol (12.5%, w/v). The soluble receptor retained the specificity of binding noted in the particulate fraction. However, Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka = 16 x 10⁹m⁻¹) for the hormone was 5 times greater than that of the particulate receptor (Ka = 3 x 10⁹m⁻¹).
The soluble prolactin receptor has been purified approximately 1500-fold by affinity chromatography using hGH-coupled N-hydroxysuccinimide ester of 3,3′-diaminodipropylaminosuccinyl agarose (Affi-Gel 10, Bio-Rad Laboratories). The receptor was labile in the presence of many of the commonly used dissociating reagents such as sodium thiocyanate, guanidine hydrochloride, and acetic acid. However, magnesium chloride (5 m) proved to be most effective for the elution of the receptor from the affinity adsorbent. About 0.5 mg of partially purified receptor protein could be obtained from 100 g of mammary gland. By gel filtration on Sepharose 6B the molecular weight of the receptor was found to be approximately 220,000. Polyacrylamide gel electrophoresis (gel concentration 7.5%, w/v; pH 8.9) of the partially purified receptor revealed several protein bands. Upon assaying the material eluted from gel slices, the receptor activity coincided with one or possibly two of the major protein bands at Rf 0.12.
These studies suggest that it is feasible to utilize the soluble receptor for the development of a very sensitive radioreceptor assay for prolactin and, further, to obtain a highly purified receptor in sufficient quantity to facilitate studies on its physiological, biochemical, and immunological properties.
A study of the variables in techniques for alkaline hydrolysis of proteins and for chromatographic analysis of the products has led to a method for the accurate determination of tryptophan. Quantitative recoveries of tryptophan are obtained when proteins (1 to 5 mg) are hydrolyzed at 110° or 135° in 0.6 ml of 4.2 n NaOH containing 25 mg of starch. The hydrolysis is performed in polypropylene liners sealed inside glass tubes evacuated to below 50 µm of mercury. Ion exchange chromatography of tryptophan on Beckman PA-35 resin (column height 8 or 12 cm) has been accomplished in 30 to 50 min with pH 5.4 buffer, 0.21 n in Na⁺.
The details in the procedure which make possible complete recovery of tryptophan include: (a) addition of the sample at pH 4.25 instead of at pH 2.2 in order to avoid loss of tryptophan in citrate buffer at acid pH; (b) the use of NaOH instead of Ba(OH)2 to avoid loss of tryptophan by adsorption on BaSO4 or BaCO3; (c) the inclusion of starch as the most effective antioxidant tested; and (d) chromatography with a buffer which separates tryptophan from Nε-(dl-2-amino-2-carboxyethyl)-l-lysine, which can be formed in significant quantities during alkaline hydrolysis. Molar calculations of protein concentrations are based on the results from analysis of an acid hydrolysate run parallel with the alkaline hydrolyses.
Integral values (100 ± 3%) have been obtained for the expected number of tryptophan residues in tryptophyl-leucine, human serum albumin, porcine pepsin, sperm whale apomyoglobin, and in bovine α-chymotrypsin, trypsin, deoxyribonuclease, and serum albumin. Since carbohydrate does not interfere, the procedure is applicable to foods and has been tested on normal and opaque-2 maize meals and on wheat flours.
This chapter discusses the assay of inorganic phosphate, total phosphate, and phosphatases. The phosphomolybdate complex is reduced by ascorbic acid. The method is about seven times as sensitive as the Fiske–SubbaRow procedure and involves less pipetting. One can easily determine 0.01 micromole of phosphate. Pyrophosphate breaks down about 5% in the method and compounds such as glucose 1-phosphate also break down somewhat, so that the method is not very satisfactory for determining inorganic phosphate if labile phosphate esters are present in large excess. The sample of organic phosphate and a drop of magnesium nitrate solution in a small test tube are taken to dryness by shaking the tube in flame. The ashing procedure is rapid and is good for various biological materials and phosphate esters such as nucleic acid, carbohydrate phosphate esters, viruses, and phospholipids. The assay method of phosphatases for inorganic phosphate can be used as an assay for phosphatases hydrolyzing stable phosphate esters such as glucose-6-phosphate, ribose-5-phosphate, and histidinol phosphate. The enzyme incubation can be stopped with the one ascorbic-molybdate solution thus avoiding an extra pipetting.
Lectins play an important role in the development of immunology. Lectins also find application in serological laboratories for typing blood and determining secretor status, separating leucocytes from erythrocytes, and agglutinating cells from blood in the preparation of plasma. They serve as reagents for the detection, isolation, and characterization of carbohydrate-containing macromolecules, including blood-group antigens. In their interaction with saccharides, lectins serve as models for carbohydrate-specific antibodies, with the important advantage to purify lectins in gram quantities. Lectins are classified according to their carbohydrate-binding specificity that includes D-mannose(D-glucose)-binding lectins and 2-acetamido-2-deoxy-D-glucose-binding lectins. The chapter considers only those lectins that have been purified to homogeneity, and studied with regard to their biophysical, biochemical, and carbohydrate-binding specificity. The chapter also describes the cell-binding and biological properties of lectins. The chapter concludes with the description of several glycopeptide structures showing the carbohydrate-binding loci with which various lectins interact.
A procedure is described to determine 2-keto-3-deoxyoctonate (KDO) present in lipopolysaccharide (LPS) of gram-negative bacteria. The method involves the treatment of LPS with 0.2 n H2SO4 at 100°C for 30 min to release KDO, followed by its reaction with periodic acid, sodium arsenite, and thiobarbituric acid. The red chromophore thus formed is kept in solution at room temperature by adding dimethylsulfoxide to the reaction mixture. The final color is stable for days at room temperature and facilitates accurate determination of KDO in microgram quantities. KDO contents of cell surface antigens and glycolipids from gram-negative bacteria are presented as illustrations of the accuracy and sensitivity of the assay.
A monovalent subunit of concanavalin A (Con A) was tested for mitogenic effects on murine splenic lymphocytes in vitro. In contrast to the effects of intact Con A, the monovalent derivative was not mitogenic at any concentration tested. Furthermore, prior exposure of splenic lymphocytes to monovalent Con A rendered the cells refractory to stimulation by the unmodified lectin.
Sucrose-dependent adherence appears to be the key element in the virulence of the cariogenic microbe, Streptococcus mutans. A number of different experiments have implicated glucans produced from sucrose by the microbe’s glucosyltransferases (GTF) as the primary factor in adherence. Therefore, it has been hypothesized that interference with either the enzyme or the glucans would be sufficient to reduce the organism’s virulence [reviewed by van Houte (1)]. This hypothesis has been at least partially validated. Treatment of viable S. mutans cells with glucanohydrolases (2–5) during glucan synthesis reduces in vitro adherence and in vivo caries incidence. Antibodies specific for partially purified GTF likewise effectively inhibit adherence of the microbe (6,7). Finally, mutants of S. mutans which are defective in glucan synthesis are neither adherent nor virulent (8,9).
The lectin present in the mucus of the snail Arion empiricorum was isolated by ion exchange chromatography. Purity was demonstrated by immunelectrophoretic analysis, immunization studies, and polyacrylamide gel electrophoresis. With the latter we found a molecular weight of 43,000. Hemagglutination inhibition studies revealed that carbohydrates play a minor role in the agglutination reaction of A. empiricorum lectin. Stronger inhibition could be achieved with human serum and the serum of several animal species. These findings were clarified by the demonstration that some serum proteins were precipitated by A. empiricorum lectin. Besides its agglutinating and precipitating properties the purified A. empiricorum lectin possesses proteinase-inhibiting properties, as demonstrated by the inhibition of casein-digestion by trypsin and plasmin.
The red alga, Agardhiella tenera was found to contain a glycoprotein which agglutinates mouse leukemia cells, L5178Y but not L1210. It also agglutinates guinea pig and rabbit erythrocytes, and has weak activity against human A, B and O, mouse, horse and sheep erythrocytes and hamster and mouse lymphocytes. The agglutination was not inhibited by simple sugars. The major active component was purified and determined to be a beta-structure protein containing 2.7% glucose as sugar moiety. The molecular weight was estimated to be 12,000 by gel filtration and 13,000 by polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. Its isoelectric point was 6.1, and it contained high amounts of glycine, serine and threonine, but no half cystine or histidine. It had no subunit structure, and the C- and N-terminal amino acids were threonine and arginine, respectively.
A mixture of isophytohemagglutinins has been isolated from the fleshy arils of the spindle tree seeds (Evonymus europaea L.) by fractional precipitation of the saline extract of the arils by (NH4)2SO4 at a 0.40% saturation. Successive preparative disc electrophoresis on polyacrylamide gel affords separation of one slower moving component, phytohemagglutinin I, from the mixture of other isophytohemagglutinins that have a very similar electrophoretic mobility. Phytohemagglutinin I has a sedimentation coefficient Sw,20 of 7.1 S and an approximate mol. wt of 127 000. Amino acid analysis shows a high amount of aspartic acid, alanine and glycine but also significant amounts of serine, threonine, cysteine and methionine. Aspartic acid is the only N-terminal amino acid found by the dansylation technique. Phytohemagglutinin I contains glucosamine and 4.7% neutral sugar. Its approximate pI in citrate/phosphate buffer is 4.4-4.5. The metal content amounts to 0.250% Ca, 0.019% Mg, 0.034% Zn and 0.026% Cu. Mn is not present. Ultracentrifugation analysis reveals homogeneity in the sedimentation behavior of the mixture of isophytohemagglutinin, an Sw,20 of 7.1 S and an approximate mol. wt of 119 000. The mixture has an amino acid composition closely resembling that of phytohemagglutinin I and an identical pI but contains only 1.9% neutral sugar. Two N-terminal amino acids were shown to be present, aspartic acid and tyrosine. With the exception of Cu which is absent, the metal content is almost the same as that of phytohemagglutinin I. Both phytohemagglutinin I and the mixture are devoid of anti-A1 activity and show detectable anti-H, anti-B and anti-A2 erythroagglutinating activity in approximate limit concentrations of 2.5, 5 and 10 mug/ml, respectively. This activity is not influenced by the presence of EDTA, Ca2+ or Mg2+, but is stimulated by Zn2+. Mn2+ and Co2+ have an inhibitory effect. None of the simple sugars tested inhibited the hemagglutination reactions.
1.1.|A new method is described for the isolation and purification of concanavalin A based on its specific adsorption on cross-linked dextran gels and subsequent displacement with d-glucose.2.2.|The yield of concanavalin A by this method is approx. 2.0–2.4 g/100 g jack bean meal. The chromatographic recovery is of the order of 94%.3.3.|Concanavalin A obtained by this procedure is approx. 98% precipitable with dextran NRRL B-1355-S.4.4.|(NH4)2SO4 fractionation of a saline extract of jack bean meal showed the maximum localization of concanavalin A activity to be in the fraction precipitated between 0.50 and 0.60 saturation of (NH4)2SO4.5.5.|The nature and specificity of binding of concanavalin A to cross-linked dextran gels have been shown to be generally similar to that of concanavalin A-polysaccharide interaction by demonstrating the reversal of the binding and subsequent elution of the protein from Sephadex G-50 with the same low molecular weight carbohydrates which inhibit concanavalin A-polysaccharide interaction.
Lectins provide new tools for studying polysaccharides, glycoproteins, and cell surfaces, and for cancer research.
The first plant agglutinin was discovered by Stillmark in 1888. It was non‐specific. Landsteiner later observed a certain degree of species specificity in some plant agglutinins, and Boyd and Renkonen found some to be blood antigen specific. Various plant hemagglutinins, called “lectins” by Boyd have a number of interesting and sometimes useful specificities: Anti‐A, anti‐A 1 , anti‐B, anti‐H, anti‐A+B, anti‐B+H, anti‐M, anti‐N, and anti‐Gy (peanut factor). No anti‐Rh lectins have thus far been found.
Even the “nonspecific” lectins have specificity in the sense that they combine with certain definitely characterized receptors on the erythrocyte, as demonstrated by inhibition experiments. This is true for example of the lectin from Rieinus communis , the first to be discovered.
Lectins are used to subdivide bloods of groups A and AB into subgroups, to diagnose secretors and nonsecretors of the ABH substances, in routine ABH blood grouping, in MN blood grouping, in the separation of A and O erythrocytes, in population studies for the “new” factor Gy, in the stimulation of mytosis in leukocytes, in the search for human blood factors in animals, in the study of the blood group substances, and in investigations of the number of specific receptors on a single erythrocyte.
The specificity of the blood‐group‐specific lectins is not always less sharp than that of naturally occurring hemagglutinins, and is sometimes sharper.
A number of workers have purified various lectins to a greater or lesser degree, usually by salting out procedures, sometimes by alcohol or acetone precipitation, sometimes done in the cold. Some lectins have been obtained crystalline.
It has been suggested that the role of lectins in the plant is the transportation and immobilization of carbohydrates, e.g . those being laid down in the seed as reserve material.
It has been shown that the presence of specific lectins in the plant is under genetic control.
It is suggested that the study of lectins may afford valuable information as to the chemical nature of the blood‐group‐specific portions of natural hemagglutinins.
Résumé
La première phytagglutinine a été découverte par Stillmark en 1888. Elle n'était pas spécifique. Par la suite, Landsteiner a observé un certain degré de sptéificité d'espèce chez certaines phytagglutinines et Boyd et Renkonen ont trouvé que certaines d'entre elles étaient spécifiques des groupes sanguins. Plusieurs phythémagglutinines, appelées «lectines» par Boyd ont un certain nombre de spécificités intéressantes qui sont parfois měme utiles: Anti‐A, anti‐A 1 , anti‐B, anti‐H, anti‐A+B, anti‐B+H, anti‐M, anti‐N, anti‐Gy (peanut factor: facteur arachide). Aucune lectine anti‐Rhésus n'a été trouvée jusqu'è ce jour.
Měme les lectines «non spécifiques» ont une spécificité par le fait qu'elles se combinent avec certains récepteurs qui se trouvent au niveau de l'érythrocyte et qui posèdent un caractère défini, ainsi que cela a pu ětre démontré par certaines épreuves d'inhibition. Ceci est le cas par exemple pour la lectine du Ricinus communis qui a été la première à ětre déconverte.
Les lectines sont utilisées pour subdiviser en sous‐groupes les groupes A et AB, pour déterminer les secréteurs et les non‐secréteurs de substances A, B, H, pour la détermination de routine des groupes ABH, pour la détermination des facteurs M et N, pour séparer les érythrocytes A des érythrocytes O, pour étudier le «nouvrau» facteur Gy dans les populations, pour stimuler la mitose chez les leucocytes, pour rechercher les facteurs sanguins humains chez les animaux, pour étudier les substances de groupes sanguins, pour rechercher le nombre de récepteurs spécifique sur un seul érythrocyte.
La spécificité des lectines spécifiques de groupe sanguin est tout aussi nette que les hémagglutinines naturelles; elle est meilleure parfois.
Un nombre considérable de chercheurs ont purifié les différentes lectines è un degré plus ou moins grand et utilisant d'ordinaire des procédés de relargage (salting out) soit par précipitations à l'alcohol ou à l'acétone, soit par le froid. Certaines lectines ont été obtenues sous forme cristallisée.
On suppose que des lectines ont dans les plantes une fonction de transport et d'imobilisation des hydrates de carbone, par exemple des hydrates de carbone qui sont déposés dans les semences comme matériel de réserve
On a pu démontrer que la présence de lectine spécifique dans les plantes est héréditaire.
On pense que l'étude des lectines peut apporter des informations vaiables sur la nature chimique de certaines parties des hémagglutinines naturelles spécifiques de groupes sanguins.
Zusammenfassung
Das erste Pflanzenagglutinin wurde 1888 von Stillmark entdeckt. Es war unspezifisch. Später bcobachtete Landsteiner bei einigen Pflanzenagglutininen eine mäßig ausgeprägte Artspezifität, und schließlich fanden Boyd und Renkonen cinige mit Blutgruppenspezifität. Zahlreiche Pflanzenagglutinine— Boyd nennt sie «Lectinen»l—zeigen eine Anzahl interessanter, gelegentlich sogar praktisch verwertbarer Spezifitäen: Anti‐A, Anti‐A 1 , Anti‐B, Anti‐H, Anti‐A+B, Anti‐B + H, Anti‐M, Anti‐N und Anti‐Gy (Erdnußfaktor). Lectine mit Anti‐Rh‐Spezifität wurden bisher nie beobachtet.
Sogar die «unspezifischen» Lectine zeigen insofern eine gewisse Spezifität, indem sie sich an gewisse definierte Rezeptoren der Erythrozytenoberfläche binden, was an Hand von Hemmversuchen bewiesen werden kann. Dies gilt z. B. für das zuerst entdeckte Pflanzenagglutinin, nämlich das Lectin von Ricinus communis .
Lectine werden für folgende Zwecke gebraucht: Unterteilung der Gruppen A und AB in Untergruppen, Ermittlung von Sekretoren und Nichtsekretoren von ABH‐Sulstanzen, Routine‐ABH‐Bestimmungen, MN‐Bestimmungen, Trennung von A und O‐Erythrozyten, Populationsstudien mit dem «neuen» Gy‐Faktor, Stimulation der Mitose bei Leukozyten, Erforschung von menschlichen Blutfaktoren bei Tieren, Erforschung der Blutgruppensubstanzen, Bestimmung der Zahl von spezifischen Rezeptoren an der Oberfläche eines einzelnen Erythrozyten.
Die Spezifität der hlutgruppenspezifischen Lectine ist nicht immer weniger ausgeprägt als die der natürlichen Hämagglutinine; gelegentlich ist sie scliärfer ausgeprägt.
Eine Reihe von Lectinen wurden mehr oder minder stark gereinigt, sei es durch Aussalzung oder durch Alkohol‐ bzw. Acetonfällung in der Kälte. Es gelang, einige Lectine zu kriutallisieren.
Es wird angenommen, daß die Lectine am Kohlenhydrattransport und deren Fixation als Resevematerial im Samen beteiligt sind.
Es wurde gezeigt, daß die Anwesenheit spezifischer Lectine in den Pflanzen von genetischen Faktoren kontrolliert wird.
Es erscheint alti wahrscheinlich, daß das Studium der Lectine wesentliche Aufschlüsse über die chemische Natur des blutgruppenspezifischen Teils der natürlichen Hämagglutinine vermitteln wird.