Manju Sarkar

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail

A sialic acid binding lectin, AchatininH, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242000, having identical subunits of Mr 15000. The lectin agglutinated rabbit erythrocytes in the presence of Ca2−. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of AchatininH. Among the inhibitors used the glycoconjugate containing α2→-6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing α2→3 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.

Identification and isolation of an agglutinin from uterus of rats

A sialic acid-binding agglutinin was purified to apparent homogeneity by affinity chromatography on fetuin-sepharose column from the rat uterine homogenate in estrus. The agglutinin is Ca++ dependent, a glycoprotein, and composed of two very closely associated bands of molecular weights 28,000 and 30,000 and pIs of 4 and 4.1. Several sialoglycoproteins, sialic acid, EDTA, glucuronic acid and heparin acted as an inhibitor of the agglutinin.

A novel galactosyl-binding lectin from the plasma of the blood clam, Anadara granosa (L) and a study of its combining site

The marine blood clam species Anadara granosa (L) belong to arcidae, a family with some extraordinary haematological features. The plasma of this species exhibited strong haemagglutinating activities, from which a galactosyl binding lectin, Anadarin P, was purified in a single step affinity chromatography using Sepharose 4B-asialofetuin as an affinity matrix. The purified lectin, eluted with lactose, was found to be homogeneous by alkaline polyacrylamide disc gels, gel-filtration and isoelectric focusing. Native Mr of the lectin was 130,000 having a PI value of 6.82 and was composed of two subunits of Mr 17,000 and Mr 16,000 which were noncovalently bound. The lectin was remarkably thermostable; the agglutinating titre remained unchanged over a wide range of pH (from 5 to 10) but increased with neuraminidase treated rabbit erythrocytes. Anadarin P combining site has been proposed to be small pocket-like structure which recognised only C-3 and C-4 hydroxyl groups of D-galactose. Presence of bulky groups at C-2 and C-6 exert strong steric hindrance as L-arabinose, 2-deoxy-D-galactose and D-xylose are better inhibitors than D-galactose. The lectin fails to differentiate methyl substituted galactosides as both α- and β- methyl galactosides are equally active; but in case of substituted phenyl glycosides, the lectin shows different affinity towards a and (3 anomers. The avidity of the lectin to bind the aromatic aglycons of galactosides suggests the presence of a hydrophobic region in the combining site. Interactions with some dissaccharides indicate the presence of an extended area near the monosaccharide binding site.

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-beta-D-galactoside, and the best inhibitor was N-acetyllactosamine.

Immobilization of antibodies on a new solid phase for use in ELISA

Antibodies to myoglobin were immobilized by covalent linkage to polyester film for use in a solid-phase ELISA. The covalent linkage of antibody to this new solid phase was accomplished by partial acid hydrolysis of the film followed by periodate oxidation. About 60 ng of protein could be immobilized per cm2 of film and the binding was stable. Antimyoglobin IgM immobilized on the films was used in the ELISA technique to detect myoglobin within the range 0.25-10 ng. The assay procedure was found to be very accurate and the coefficient of variation of each concentration ranged from 0.63 to 2.1%. Furthermore the immobilized film could be re-used after dissociating the antigen antibody complexes.

N-glycolylneuraminic acid specific lectin from Pila globosa snail.

A N-glycolylneuraminic acid-specific lectin (PAL) has been purified from an albumin gland extract of the apple snail, Pila globosa. Purification is conducted on a bovine submaxillary mucin-Sepharose 4B affinity matrix followed by gel filtration on a Sepharose 6B column. The lectin agglutinates rabbit erythrocytes. The hemagglutination activity is dependent on Ca2+ concentration in a significant manner but with a remarkable behaviour. The lectin is a trimeric glycoprotein of native Mr 440 kDa with 25% carbohydrate and is composed of three nonidentical subunits of molecular weights 190, 145, and 105 kDa. It has a pI of 7.0. The lectin exhibits a unique and strict specificity toward N-glycolylneuraminic acid and this phenomenon discriminates it from other known sialic acid binding lectins. The uniqueness indicates the absolute need for a glycolyl substitution on the amino residue and of a glyceryl side chain on the exocyclic part and an axial -COOH group in neuraminic acid. The presence of an acetyl substitution on the exocyclic part impedes lectin-ligand interaction.

A galactose specific agglutinin from the hemolymph of the snail Achatina fulica: Purification and characterization

A galactose specific agglutinin has been detected in the hemolymph of the snail Achatina fulica fulica Bowdich. The agglutinin, which agglutinates rabbit erythrocytes, has been purified by affinity chromatography on crosslinked guargum followed by gel filtration on a Biogel P-200 column. The purified agglutinin has a native molecular weight of 210,000 and comprises of non covalently linked identical subunits of molecular weight 15,000. The pI of the agglutinin was found to be 8.1. The activity of the agglutinin is independent of divalent cations. The inhibition of hemagglutination data reveals that 2-deoxy-D-galactose is the most potent inhibitor of the agglutinin followed by galactose and lactose.

Estradiol benzoate induced changes in protein synthesis and lysosomal hydrolases in the pituitary of male rats.

In vitro protein synthesis, lysosomal hydrolases activity and peroxidase activity in the anterior pituitary were estimated in adult male rats treated with 50 micrograms of estradiol benzoate (EB) for 1 day or 7 days. Pituitary protein synthesis, protein and RNA content increased after 7 days. A significant increase in total and membrane-bound acid phosphatase was noted after 1 day or 7 days of EB treatment whereas total beta-glucuronidase activity decreased in both 1 and 7 day group. Cathepsin activity increased after 7 days and pituitary peroxidase system did not change by EB treatment. These findings suggest that immediate change in the enzyme milieu may be one of the first reactions by which EB expresses its feedback control.

Purification and characterization of an agglutinin from mucus of the snail Achatina fulica

The mucus of the snail Achatina fulica shows the presence of an agglutinin that nonspecifically agglutinates human erythrocytes. The agglutinin has been purified by affinity chromatography using Sepharose 4B-hog gastric mucin as the affinity matrix. Homogeneity was checked by polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and gel filtration. The agglutinin is a glycoprotein of native molecular weight 70,000. The isoelectric point of the protein was found to be 8.0. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 32% of the total amino acid residues. The agglutinin has 10% carbohydrate (wt/wt) and the most abundant sugar is N-acetylglucosamine. The cd spectra of the agglutinin show the presence of random coil conformation. The inhibition of hemagglutination data indicates that the agglutinin is specific for beta glycosides of D-Gal and D-GalNAc.

Homogeneous enzyme immunoassay of diosgenin and its glycosides

Homogeneous enzyme immunoassay has been used as a tool for the determination of diosgenin and its glycosides in plants. Diosgenin antisera was found to inhibit the activity of diosgenin hemisuccinate-horseradish peroxidase conjugate which was reversed by the addition of free diosgenin or its glycosides. The increase of enzyme activity was proportional to the quantity of the hapten over a certain range of hapten concentration. Thus, a minimum of 2.5 micrograms/ml of diosgenin and 11.5 micrograms/ml of diosgenin glycosides could be determined by this method. The results were comparable with those obtained by high-performance liquid chromatography and gravimetric methods.