A New Lectin from Meadow Saffron (Colchicum automnale)

A lectin has been isolated from tubers of the meadow saffron (Colchicum autumnale). It is an octameric protein (M_r 100,000) composed of 4A- and 4B-subunits of M_r 15,000 and 10,000, respectively. It is a glycoprotein with 4.4% carbohydrate, the main sugars are (N-acetyl-) glucosamine, mannose, fucose, and xylose. Although the Colchicum autumnale agglutinin (CAA) agglutinates human red blood cells, it has a much higher activity with rabbit erythrocytes. With respect to its carbohydrate-binding specificity CAA behaves rather unusually as it is inhibited by lactose, galactose, N-acetylgalactosamine and related sugars when assayed with human red blood cells but not in assays with rabbit erythrocytes.     

A new agglutinating activity from wheat flour inhibited by tryptophan

A new compound endowed with agglutinating activity, designated the flour agglutinin, was extracted from wheat flour with water and purified by gel filtration and ion-exchange chromatography. The haptenic inhibitors of the plant agglutinis do not affect flour agglutinin activity which, on the other hand, is inhibited by d- and l-tryptophan.Flour agglutinin has a molecular weight of about 5 · 10⁴ as determined by gel filtration. It consists of a neutral heteropolysaccharide constituted of d-xylose and l-arabinose, and is homogeneous as judged by sedimentation analysis. Flour agglutinin activity is destroyed by treatment with Cellulase 2000 and periodate, but is not affected by α-amylase and proteolytic enzymes.Compared to germ agglutinin, flour agglutinin exhibits a peculiar range of cell specificity. It agglutinates several normal cell types, but has no effects on some neoplastic cells tested. Tryptic digestion of erythrocytes does not affect their susceptibility to flour agglutinin-induced agglutination.     

Ultrastructural and histological study of degenerative changes leading to black gills in grass shrimp exposed to a dithiocarbamate biocide

The presence of a carbohydrate-specific opsonin, distinguishable from hemolymph agglutinin, was demonstrated in the American lobster, Homarus americanus. Hemolymph opsonin, measured by the enhancement of hemocyte phagocytosis of sheep erythrocytes, was found to be more heat and acid labile than the agglutinin. Both opsonization and agglutination of sheep erythrocytes were inhibited by monosaccharides; however, maximal effects on opsonization were observed with d-(+)mannose, which did not affect agglutination. On the other hand, N-acetyl-d-glucosamine significantly inhibited agglutination but had little effect on opsonization. Fractionation of the molecules was accomplished by differential adsorption to Sephadex G-200 using a 0.15 m NaCl buffer. Hemolymph recovered in the column effluent was enriched for opsonic activity and devoid of agglutinin. However, both opsonin and agglutinin could be detected in the effluent when the column was equilibrated with a 0.48 m NaCl buffer. Neither agglutinin nor opsonin were able to pass an ultrafiltration membrane capable of retaining molecules greater than 3 × 10⁵ daltons.     

Lectin-Like Constituents of Foods Which React with Components of Serum, Saliva, and Streptococcus mutans

Hot and cold aqueous extracts were prepared from 22 commonly ingested fruits, vegetables, and seeds. When tested by agar diffusion, extracts from 13 and 10 of the foods formed precipitin bands with samples of normal rabbit serum and human saliva, respectively; extracts from four of the foods also reacted with antigen extracts of strains of Streptococcus mutans. When added to rabbit antiserum, extracts from 18 of 21 foods tested inhibited reactivity with antigen extracts derived from S. mutans MT3. Extracts from 16 foods agglutinated whole S. mutans cells, whereas those from 10 foods agglutinated human erythrocytes of blood types A and B. The lectin-like activities of extracts which reacted with human saliva were studied further. Pretreatment of saliva-coated hydroxyapatite (S-HA) beads with extracts of bananas, coconuts, carrots, alfalfa, and sunflower seeds markedly reduced the subsequent adsorption of S. mutans MT3. Pretreatment of S-HA with banana extract also strongly inhibited adsorption of S. mutans H12 and S. sanguis C1, but it had little effect on attachment of Actinomyces naeslundii L13 or A. viscosus LY7. Absorption experiments indicated that the component(s) in banana extract responsible for inhibiting streptococcal adsorption to S-HA was identical to that which bound to human erythrocytes. The banana hemagglutinin exhibited highest activity between pH 7 and 8, and it was inhibited by high concentrations of glucosamine, galactosamine, and, to a lesser extent, mannosamine. Other sugars tested had no effect. The selective bacterial adsorption-inhibiting effect noted for banana extract was also observed in studies with purified lectins. Thus, pretreating S-HA with wheat germ agglutinin and concanavalin A inhibited adsorption of S. mutans MT3 cells, whereas peanut agglutinin, Ulex agglutinin, Dolichos agglutinin, and soybean agglutinin had little effect; none of these lectins affected attachment of A. viscosus LY7. Collectively, the observations suggest that many foods contain lectins which can interact with components of human saliva and S. mutans cells. Because of their potential to influence host-parasite interactions in the mouth and elsewhere in the gastrointestinal canal, these reactions warrant further study.     

A galactose specific agglutinin, a blood-group H active polysaccharide, and a trypsin inhibitor in albumin glands and eggs of Arianta arbustorum (Helicidae).

Eggs and albumin glands of the land snail Arianta arbustorum contain a powerful agglutinin which reacts specially with rabbit erythrocytes. The agglutination can be inhibited completely by di-, tri-, and oligosaccharides with α-glycosidically (1 → 6) bound galactose residues. β-Linked sugars do not inhibit the agglutinin. The agglutinin activity is not dependent on Ca²⁺ ions. Eggs and albumin glands also contain a blood-group active polysaccharide which, unlike the polysaccharide from the albumin gland of Helix pomatia (Baldo, B. A., and Uhlenbruck, G. 1973. Cross-reactive human blood group H-active polysaccharide from Helix pomatia. I. Detection with catfish anti-H and eel sera. Immunology, 25, 1–13) does not react with anti-H_eel, but does react with the agglutinins of Evonymus europaeus and Laburnum alpinum. The Arianta polysaccharide has been purified and shown to be galactogen. Finally, the occurrrence of a strong trypsin inhibitor has been demonstrated in the extracts of eggs and albumin glands. The inhibitor has been separated by column chromatography. The precipitation lines of both substances have been identified in the immunoelectrophoretogram of the extracts of albumin glands and eggs.     

Ovarian infection and fungal spore oviposition in the blackfly Prosimulium mixtum

In order to ascertain whether agglutinins can serve as links to bind hemocytes of Helix pomatia to mammalian erythrocytes, rosette-formation tests were performed. These involved pretreatment of H. pomatia hemocytes with each of 15 nonnative agglutinins and incubation of them with human erythrocytes. It has been found that, of the agglutinins tested, wheat germ agglutinin (WGA) as well as those from Ricinus communis, Axinella polypoides, Anguilla anguilla (anti-H_eet), concanavalin A, and Limulus polyphemus caused rosette formation with human erythrocytes. In addition, it has been found that a small number of H. pomatia hemocytes are capable of direct binding to erythrocytes of mice, rabbits, rats, and sheep.     

Lectin-like activity from Persea americana

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.     

Partial characterization of a natural agglutinin in the hemolymph of the lobster, Homarus americanus

Partial characterization of a natural agglutinin in the hemolymph of the lobster showed: (1) Calcium ion stabilized the agglutinin against inactivation at moderate (35–45°C) temperatures as well as against a reversible increase in activity at nonphysiological pH levels (pH 6 and 9). (2) Serum frozen at ?20°C in the absence of calcium showed an increase in agglutinin activity. (3) Heat inactivation curves indicated a single component for the agglutinin. (4) Heat inactivation destroyed the ability of the agglutinin to be adsorbed to the erythrocyte antigen. (5) The chemical nature of the antigenic reactive site differed with the species of erythrocyte tested; D-glucosamine blocked or inhibited adsorption of the agglutinin to erythrocytes of most species tested. (6) In vivo the agglutinin is located in the hemocytes.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Root Tissue Response of Two Related Soybean Cultivars to Infection by Lectin-treated Meloidogyne spp.

Treatment of second-stage juveniles (J2) of Meloidogyne incognita race 1 and M. javanica with soybean agglutinin, Concanavalin A, wheat germ agglutinin, Lotus tetragonolobus agglutinin, or Limax flavus agglutinin or the corresponding competitive sugars for each of these lectins did not alter normal root tissue response of soybean cultivars Centennial and Pickett 71 to infection by M. incognita race 1 or M. javanica. Giant cells were frequently induced in Centennial and Pickett 71 roots 5 and 20 days after inoculation of roots with untreated J2 of a population of M. incognita race 3. Treatment of J2 of M. incognita race 3 with the lectins or carbohydrates listed above caused Centennial, but not Pickett 71, root tissue to respond in a hypersensitive manner to infection by M. incognita race 3. Penetration of soybean roots by J2 of Meloidogyne spp. was strongly inhibited in the presence of 0.1 M sialic acid. Treatment of J2 with sialic acid was not lethal to nematodes, and the inhibitory activity of sialic acid was apparently not caused by low pH. These results suggest that carbohydrates may influence plant-nematode interactions.     

Immunochemical studies of organ and tumor lipids XVIII. Cytolipin R determinants in the rat erythrocyte membrane

Summary Rabbit antisera to rat lymphosarcoma contain antibodies that agglutinate trypsinized rat erythrocytes. These reactions can be specifically inhibited by cytolipin R, a ceramide tetrasaccharide isolated from rat lymphosarcoma. The agglutinin in the rabbit antisera can be absorbed with untreated erythrocytes, showing that cytolipin R determinants are present in the intact rat erythrocyte membrane. Untreated erythrocytes are able to react with antibody, but presumably the number of cytolipin R determinants necessary for agglutination becomes available only after treatment with trypsin. The anti-cytolipin R antibodies in anti-rat lymphosarcoma sera that cause hemagglutination and those that fix complement with this hapten are different, since the agglutinin can be absorbed completely without appreciable decrease in complement-fixing antibody.A preliminary report was presented at the 53rd Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April, 1969 (1969,Fed. Proc. 28:700).     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

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.     

A new cold agglutinin from Achatina fulica snails

An electrophoretically homogeneous agglutinin was purified from the albumin gland of Achatina fulica snails using asialofetunin-Sepharose 4B as an affinity column. The agglutinin was found to be temperature sensitive; it agglutinated rabbit and human umbilical cord erythrocytes only at low temperature. It was found to be specific for methyl-β-d-galactoside, and the best inhibitor was N-acetyllactosamine.     

5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

A development-dependent hemagglutinin from cucumber surfaces: I. Hemagglutinin activity of cotyledon surfaces

During studies on the chemistry of plant surfaces, we observed that concomitant with the development of cucumber (Cucumis sativus L. cv `ELEM') cotyledons an agglutinin that agglutinates human erythrocytes appeared on epicuticular surfaces. The agglutinin was released from cotyledon surfaces into distilled water by a 1-minute immersion (or even less). Homogenization of the washed cotyledons released residual agglutinating activity. The surface-located hemagglutinating activity was age-dependent and occurred in dark- and light-grown cotyledons. Agglutinating activities were present in light-grown cotyledons of 2- to 14-day-old seedlings and in dark-grown cotyledons of 3- to 17-day-old seedlings. Agglutinating activities were maximal in cotyledons of 3- to 4-day-old seedlings. No activity could be detected in dry seeds or in seedlings up to 2 days and after 17 days of germination. The hemagglutinating activity was specifically inhibited by N,N′,N″-triacetylchitotriose, chitin, and chitosan.     

Immunological studies of the mechanism of anaemia in experimental Trypanosama evansi infection in rats

Pathological changes in the blood of rats acutely infected with Trypanosoma evansi and the probable mechanism of the accompanying anaemia, were investigated. A severe anaemia, together with rreticulocytosis and hepato-splenomegaly, were regularly observed. Histological examination of the liver, spleen and bone-marrow confirmed the increasedin erythropoietic activity that the observed anaemia was due to increasedextravascular destruction of erythrocytes rather than by inhibition of haemopoietic activity. All the infected rats showed significant immune responses to the infecting trypanosome peak agglutinin titres occurring 10–12 days after injection, coincidentally with maximun destruction of erythrocytes. Serological examination of sera and erythrocytes from all infected and control rats did not reveal the presence of either circulating or adsorbed erythrocyte auto--antibodies. Furthermore, there was no in vivo trypanosomal antigen coating of the erythrocytes from either infected or multiple antigen-injected rats. Repeated intraveoous injections into rats of more than 100 μg per g body weight of soluble T. evansi antigen resulted in moderately severe, probably antibody-mediated, haemolytic anaemia. It is considered that an immunologically-mediated mechanism may be responsible for the development of the anaemia accompanying T. evansi infection.     

Purification and characterization of deoxyuridine triphosphate nucleotidohydrolase from anemic rat spleen: A trimer composition of the enzyme protein

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) was purified to near homogeneity from the spleens of rats made anemic by phenylhydrazine injection; the enzyme activity in these spleens was about 30 times higher than that in spleens of untreated rats. The purified enzyme preparation showed an apparent molecular weight of 58,500 and appeared to consist of three identical subunits each with a molecular weight of about 19,500. The purified enzyme catalyzed specifically the hydrolysis of dUTP, and no other naturally occurring nucleoside triphosphates could be hydrolyzed by this enzyme. The K_m value for dUTP was 12 μm. Enzyme activity was inhibited by the addition of EDTA, whereas the enzyme preparation exhibited activity in the absence of added divalent cations. Activity was not affected by the addition of fluoride ion.     

Screening and preliminary characterization of hemagglutinins in Vietnamese marine algae

Extracts from 44 species of Vietnamese marine algae, including 15 Chlorophyta, 18 Rhodophyta and 11 Phaeophyta species, were examined for hemagglutination activity with a variety of different animal and human erythrocytes that were untreated or treated with enzymes. Almost all extracts showed activity toward at least one type of erythrocytes, although those from three Chlorophyta and two Rhodophyta species showed no hemagglutination with any type of erythrocytes examined. Strong activity was detected in extracts from two Chlorophyta (Anadyomene plicata and Avrainvillea erecta) and four Rhodophyta species (Gracilaria eucheumatoides, Gracilaria salicornia, Kappaphycus alvarezii, and Kappaphycus striatum) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of seven Chlorophyta and eight Rhodophyta species were examined for sugar-binding specificity, pH- and temperature-stability, and divalent cation-independency of hemagglutination using ammonium sulfate-precipitates prepared from their extracts. In a hemagglutination-inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides, except the Codium arabicum and Gracilaria euchematoides hemagglutinins, whose activities were inhibited by both N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. On the other hand, all of the hemagglutinins activities were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The activities of these algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations. These results indicate that Vietnamese marine algae are a good source of novel and useful lectins.