B.K. Bachhawat

PURIFICATION AND PROPERTIES OF BRAIN ALKALINE PHOSPHATASE

Abstract— 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.

Targeting of liposomes towards different cell types of rat liver through the involvement of liposomal surface glycosides

Studies on the uptake of liposomes by isolated cell types of rat liver after in vivo administration reveal that hepatocytes are three times more efficient than nonparenchymal cells in taking up liposomes having β-galactoside on their surface whereas α-mannoside liposomes are taken up preferentially by nonparenchymal cells. Nonsugar liposomes are taken up by both cells. Glycoside-containing liposomes are also cleared from the circulation at a faster rate than nonsugar liposomes. Asialofetuin and mannan inhibit both the clearance and the uptake by isolated cells of β-Gal and α-Man liposomes, respectively. These findings show that surface β-galactoside and α-mannoside can mediate selective targeting of liposomes toward parenchymal and nonparenchymal cells, respectively, of rat liver.

Monosialoganglioside liposome-entrapped enzyme uptake by hepatic cells

Monosialoganglioside liposomes are rapidly taken up by the liver as compared to dicetylphosphate, phosphatidic acid or neutral liposomes. Asialoganglioside GM 1 liposomes are taken up with the same avidity as ganglioside GM 1 liposomes. Competition experiments with asialofetuin suggest that this uptake is mediated by specific recognition of the terminal galactose residues of the glyco-lipid liposomes by the receptor present on the plasma membrane of the parenchymal cells of liver. Thus liposomes containing glycolipids with terminal beta-galactosyl residues should provide an approach for specifically directing biologically active molecules to liver parenchymal cells.

Affinity chromatography of galactose containing biopolymers using covalently coupled Ricinus communis lectin to Sepharose 4B.

A galactose-specific lectin isolated from Ricinus communis beans has been covalently coupled to Sepharose 4B activated with cyanogen bromide. The immobilized lectin retains its polysaccharide-binding property. The Sepharose-lectin can be used for the purification of polysaccharides containing terminal nonreducing galactose. Only a small fraction of native fetuin and native ceruloplasmin are retarded on Sepharose-lectin. On analysis it was observed that they had a lower content of sialic acids as compared to the native and unbound glycoproteins (sialated fractions). However, on desialation, fetuin and ceruloplasmin were completely adsorbed to Sepharose-lectin. The asialoglycoproteins interact strongly with Sepharose-lectin as compared to partially sialated glycoproteins. This has been attributed to the exposure of galactose residues of these glycoproteins on enzymatic desialation. These experiments demonstrated that Sepharose-lectin interacts with glycoproteins through their terminal, non-reducing galactose. On the basis of these experiments it is suggested that Sepharose-lectin can be used as an analytical tool for separation of fully sialated glycoproteins from the partially sialated glycoproteins.

Interaction between Concanavalin A and brain lysosomal acid hydrolases

Abstract All the five sheep brain lysosomal acid hydrolases tested, namely arylsulphatase A, acid phosphatase, β-N- acetylhexosaminidase , β-galactosidase and β-glucuronidase bind with Concanavalin A and form enzymatically active precipitate. This enzyme-Concanavalin A complex can be dissociated by α-methyl- d -glucoside and the dissociation is pH dependent; except for arylsulphatase A which dissociates maximally at pH 9.0, all other enzymes dissociate maximally at pH 4.0. The enzyme-Concanavalin A complex formation is inhibited by α-methyl- d -glucoside. Using the differential dissociation of the enzyme-Concanavalin A complex, these enzymes have been purified to the extent of 30–180-fold over the soluble lysosomal fraction. The dissociation of the enzyme-Concanavalin A complex and the inhibition of its formation by α-methyl- d -glucoside suggest the glycoprotein nature of the enzymes.

Increased circulatory half-life of liposomes after conjunction with dextran

Dextran was covalently coupled to neutral unilamellar liposomes. Dextran conjugated liposomes were cleared from the circulation at a much slower rate than unconjugated liposomes. The uptake of dextran conjugated liposomes by liver and spleen was also decreased. The amount of dextran on the surface of liposomes was found to be a determining factor for their stability in circulation. Dextran conjugated liposomes therefore may be a more effective way of controlled drug release

Synthetic glycolipids: Interaction with galactose-binding lectin and hepatic cells

Abstract Lactosyl- and melibiosyl-phosphatidylethanolamine prepared by reductive animation with sodium cyanoborohydride were incorporated into small unilamellar liposomes. Lactosyland melibiosyl-phosphatidylethanolamine liposomes are aggregated by Ricinus communis agglutinin whereas Banderiaea simplicifolia isolectin I aggregates only melibiosyl-phosphatidylethanolamine liposomes. The association constant (Ka) values of interactions of R. communis agglutinin and glycolipids were 5 × 105 and 1.2 × 105 m −1 for lactosyl- and melibiosyl-phosphatidylethanolamine, respectively, whereas the Ka for the interaction of B. simplicifolia isolectin I for melibiosyl-phosphatidylethanolamine was found to be 6 × 105 m −1. The rates of aggregation of these liposomes are strikingly influenced by the amount of glycolipid incorporated into them. In vivo studies indicate that lactosyl-phosphatidyl-ethanolamine-containing liposomes are rapidly taken up by hepatic cells due to binding of their β- d -galactopyranosyl residues by the hepatic galactose-binding lectin.

The effect of lipid composition on liposome-lectin interaction

Abstract Binding of a galactose-specific lectins from Ricinus communis (RCA1) to liposome containing gangliosides, galactocerebroside and cytolipin H reveal that the lectin binds to glycolipids containing terminal nonreducing galactose residues. Lectin binding to galactocerebroside and cytolipin is strongly influenced by the chain length of fatty acid in phospholipids used for preparing liposomes and the concentration of cholesterol in liposomes.

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 → 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 × 103m−1.

The effect of surface charges of liposomes in immunopotentiation

The purpose of this study was to establish the effect of surface charges of liposomes on its adjuvant activity to an entrapped protein antigen. The immune responses of rabbits immunized subcutaneously with lysozyme entrapped in neutraJ negatively and positively charged liposomes and compared with complete Freunds adjuvant (CFA), showed positively charged liposomes to be a better adjuvant than neutral, negatively charged liposomes and even CFA. This was true for solid liposomes also. Interestingly, injection of positively charged liposomes led to the formation of granulomas at the sites of immunization, which was not observed with neutral and negatively charged liposomes.