Purification and properties of brain alkaline phosphatase

Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion-exchange chromatography on DEAE-cellulose, and on DEAE-Sephadex followed by Sephadex G-200 filtration. By these steps, the enzyme is purified 22,920-fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p-nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described

Fluorescence studies on the interaction of some ligands with carcinoscorpin, the sialic acid specific lectin, from the horseshoe crab, Carcinoscorpius rotundacauda

The binding affinities of some ligands towards the sialic acid-specific lectin carcinoscorpin, from hemolymph of the horseshoe crab Carcinoscorpius rotundacauda have been determined by protein fluorescence quenching in presence of ligands. Among the ligands studied, the disaccharide O-(N-acetylneuraminyl)-(2→6)-2-acetamido-2-deoxy-D-galactitol has the highest Ka (l.15 × 106 M−1) for carcinoscorpin. Studies on the effect of pH on Ka values of disaccharide suggests the possible involvement of amino acid residues having pKa values around 6.0 and 9.0 in the binding activity of carcinoscorpin. There were distinct changes in the accessibility of the fluorescent tryptophan residues of carcinoscorpin by ligand-binding as checked through potassium iodide quenching

Fractionation of sialoglycoproteins on an immobilized sialic acid-binding lectin

Carcinoscorpin, the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda, has been immobilized using Sepharose. The immobilized lectin is shown to resolve the isoenzymes of alkaline phosphatase from sheep brain based on the difference in their sialic acid contents. The noninic detergent Triton X-100 does not interfere with the binding efficiency of the column up to a concentration of 1% when tested with <SUP>125</SUP>I-labeled fetuin. However, the use of hexadecyltrimethylammonium bromide produced a 45% inhibition of binding of the labeled fetuin. The efficiency of various saccharides to elute the bound fetuin from the matrix was determined. O-(N-Acetylneuraminyl) (2 &#8594; 6) 2-acetamido-2-deoxygalactitol was shown to be the most powerful agent in competing with the fetuin-lectin interaction. Moreover, D-glucuronic acid was also found to elute the bound fetuin from the immobilized lectin. <SUP>125</SUP>I-Labeled fetuin in which an artificial heterogeneity is created by partial desialylation with neuraminidase resolved into three peaks using the immobilized lectin and a gradient of the disaccharide. It is suggested that this immobilized lectin could be used in the purification and resolution of minute amounts of several sialoglycoproteins

Further characterization of the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, named carcinoscorpin, has been isolated from the horseshoe crab Carcinoscorpius rotunda cauda. It is a glycoprotein of molecular-weight 420,000, having two subunits of molecular weight 27,000 and 28,000, both subunits responding to glycoprotein stain. Leucine was detected as the only NH2-terminal amino acid. The sedimentation constant of the native lectin was found to be 12.7 s. On digestion with trypsin, the lectin gave 18 soluble tryptic peptides. This lectin was found to be antigenically unrelated to another sialic acid-binding lectin, limulin, isolated from the horseshoe crab Limulus polyphemus. A lectin-specific disaccharide alcohol namely O-(N-acetylneuraminyl) (2 &#8594; 6)2-acetamido-2-deoxy-D-galactitol was found to quench the typical tryptophan fluorescence of the native lectin at 332 nm. The association constant for this interaction was determined spectrofluorimetrically and found to be 1.82 &#215; 103 M-1

Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

The sialic acid binding loctin carcinoscorpin agglutinates Escharichia coli K12 and Salmonella minnesots R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. Vibrio cholerae, a Gram negative bacterium devoid of 2-keto-3-deoxyoctonate and Staphylococcus sureus a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoe crab

Studies on the interaction of concanavalin A with glycoproteins

Lectins (phytohaemagglutinin) are known to have the unique property of binding with certain specific sugars, polysaccharides and glycoproteins. Although the kinetics of interaction between lectins and sugar have been extensively studied, the binding characteristics of the lectins with various glycoproteins are not well understood. In this laboratory a systematic study has been initiated in relation to the interaction of lectins with glycoproteins. Concanavalin A is known to bind alpha-glucosides, mannosides and biopolymers having these sugar configurations. A galactose binding protein from caster bean has been purified to homogeneity and was found to contain mannose. This lectin was used as the source of glycoprotein for studying its interaction with concanavalin A. This study showed that the interaction is temperature dependent and the dissociation is time and alpha-methyl glucoside concentration dependent. This has led to speculate a model for cell-lectin interaction. Using concanavalin A it has been shown that all the lysosomal enzymes from brain studied were glycoprotein in nature. Moreover, using Sepharose-bound concanavalin A it has been possible to devise a method by which these lysosomal enzymes could be purified considerably. With the knowledge that the interaction between lectin and glycoprotein is not only dependent on the specific sugar present in the glycoprotein, but also on the nature of the glycoprotein it was possible to develop a novel method for immobilizing various glycoprotein enzymes, such as arylsulphatase A, hyaluronidase and glucose oxidase

PARTE II: SITUACIÓN DE LAS UNIVERSIDADES ARGENTINAS Y LATINOAMERICANAS EN RELACIÓN AL MATERIAL CADAVÉRICO PARA LA ENSEÑANZA DE LA ANATOMÍA. Part II: Argentine and Latinamerican universities situation in relation to the cadaveric material for Anatomy teaching

Para esta segunda parte se realizó una amplia invitación. Publicamos los aportes de destacados autores cuya generosa contribución al Debate lo jerarquiza. Las preguntas enviadas por el Dr. Thambi Dorai fueron enviadas a los participantes del Debate inicial y sus respuestas incluidas en el presente artículo.   A wide spread invitation was distributed for this second part. We are publishing some generous contributions from relevant authors which hierarchized the Debate. Questions sent by Dr. Thambi Dorai were envied to all the participants in the initial Debate and their answers included in the current article