D-Glucosyltransferase of Streptococcus mutans: isolation of two forms of the enzyme that bind to insoluble dextran

The D-glucosyltransferase from Streptococcus mutans 6715 has been separated into three enzymic fractions that differ in their binding to dextran and in their synthesis of dextran from sucrose. One enzymic fraction (AFF-I) does not bind to insoluble dextran, and it produces an insoluble D-glucan. Fraction AFF-IIU was eluted from a dextran affinity-column by either dextran or urea, whereas fraction AFF-IID was eluted only by dextran. Both of these fractions produce insoluble D-glucans from sucrose.     

The effect of isotype and the J kappa region on antigen binding and idiotype expression by antibodies binding alpha (1----6) dextran

Gene transfection and expression techniques have been used to produce three antibodies specific for alpha (1----6) linked dextran B512 with altered isotypes and J kappa regions. Expression of the L chain V region joined to J kappa 4 or J kappa 5 instead of to J kappa 2 reduced or abolished dextran binding. One antidextran with a reduced binding constant for dextran had the same combining site size as the parental mAb. Transfectoma Ig unreactive with dextran B512 did not bind to other class I or class II dextrans. Antibodies with J kappa 4-containing L chains expressed the 10.16.1 (anti-alpha(1----6) dextran) idiotype. In contrast variants expressing L chains with J kappa 5 lost idiotype expression, except when oligosaccharide is present on VH; all antibodies with J kappa 5 L chains continued to bind dextran but with reduced affinity. The presence of carbohydrate in VH may alter the conformation of both paratope and idiotope. Alteration of H chain isotype did not appear to significantly alter the ability of the antidextran to bind Ag; an exception may be that switching V regions to the IgM C region may decrease the apparent affinity for Ag.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion

The binding of dextran sulfate to phospholipid liposomes was investigated by microelectrophoresis experiments. The polyanion binds to neutral phospholipid liposomes (DMPC and PE) only in the presence of Ca2+. If positively charged stearylamine is incorporated in the vesicles dextran sulfate is bound without Ca2+. Negatively charged phospholipids as PS do not bind dextran sulfate, even in the presence of millimolar concentrations of Ca2+. The adsorption of dextran sulfate results in an aggregation of vesicles due to a bridging mechanism. In all cases the aggregation is followed by a disaggregation toward higher dextran sulfate concentrations. The disaggregation process starts at polymer concentrations smaller than the concentration of the onset of saturation of the adsorption. By use of the probe dilution method a fusion of small DMPC and DMPC/PE vesicles in the presence of Ca2+ and dextran sulfate was found.     

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.     

Heparin enhances the rate of binding of fibronectin to collagen.

125I-labelled fibronectin is shown to bind to both native and denatured collagen immobilized on Sephadex beads in reactions that exhibit different kinetics. The rates of both reactions were enhanced by the presence of heparin or highly sulphated dextran sulphate but not by other glycosaminoglycans or dextran sulphates having low sulphate contents.     

Integration of ion exchange and ultrafiltration steps studied during purification of formate dehydrogenase using DEAE dextran

Summary Purification of formate dehydrogenase based on ionic properties combined with ultrafiltration is described. The enzyme was allowed to bind to high molecular weight DEAE dextran at low ionic strength forming a complex of high molecular weight retained behind an ultrafiltration membrane with a cut-off range of 300 000 dalton. When the ionic strength was increased, the enzyme dissociated from the DEAE dextran and could be recovered in the filtrate.     

Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro

Washed ejaculated boar sperm and sperm from the cauda epididymis bind to the zona pellucida of fixed porcine eggs in large numbers. Sperm incubated in the presence of dextran sulfate (8 K daltons or 500 K daltons) or fucoidan and then washed no longer bind to eggs. Other acid carbohydrates (heparin, chondroitin sulfates, inositol hexasulfate, carboxymethylcellulose) fail to block sperm-egg binding even when added directly to sperm-egg suspensions. Seminal plasma and the seminal vesicle secretion contain basic proteins which bind tightly to sperm and bind reversibly to eggs preventing sperm from binding to eggs. When dextran sulfate or fucoidan are mixed with the vesicular secretion, from which seminal plasma basic proteins originate (Hunt et al., '83), the secretion loses the capacity to prevent sperm from binding to eggs; this suggests that seminal vesicle proteins can bind to the same site on zonae as do sperm and thus seminal plasma may modify sperm-egg interactions. Corpus and cauda epididymal sperm also bind in large numbers to the zona pellucida of isolated eggs but high concentrations of caput sperm, which exhibit high motility in the presence of caffeine, bind only in few numbers. Thus a component that enhances sperm-zona binding is apparently formed on the plasma membranes of uncapacitated sperm during passage through the epididymis. This finding, and an earlier observation that antibodies raised against uncapacitated sperm plasma membranes block sperm-egg binding in vivo (Peterson et al., '83) suggest that this component may be involved in sperm zona interaction in vivo.     

Changes in Lectin Activity in Plants Treated with Resistance Inducers

We studied the changes in lectin activity in tobacco leaf discs and potato tubers treated with polysaccharides (chitosan, glucomannan, and dextran sulfate), enzymes (cellulase and pectinase), or monosaccharides (glucose and glucosamine). All these substances changed lectin activity to a certain extent (significantly or as a tendency). The content of membrane lectins in the chloroplasts (tobacco leaf discs) usually decreased considerably immediately after the treatment (1–2 days) but increased later (2–4 days). Generally, a higher lectin activity was characteristic of potato tubers treated with the inducers. The enzymes increased lectin activity during the whole observation period (5 days). A pronounced antiviral activity was observed in the hypersensitive tobacco–tobacco mosaic virus system only after treatment with chitosan and glucomannan.     

Effect of Diethylaminoethyl Dextran on the Growth of Mycoplasma in Agar

The growth of certain strains of Mycoplasma is inhibited by substances present in commercial agar preparations. The addition of diethylaminoethyl (DEAE) dextran (10 mg per 100 ml) to agar media appears to enhance the growth of some strains. Of eight strains initially tested, the presence of DEAE dextran grossly enhanced the growth of three strains. One strain appeared not to be affected, and a clearly enhancing effect was not evident with four strains. Quantitative studies revealed that growth enhancement varied from 10 colony-forming units (CFU) for M. hominis type II (strain Campo) to 10(3.3) CFU for M. pulmonis (strain 880). The growth-enhancing effect is probably due to the ability of DEAE dextran to bind the sulfated polysaccharide moieties in agar and not to the DEAE dextran, per se.     

Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column

Babesia bigemina-infacted erythrocytes preferentially bind to dextran sulphate affinity columns. Subsequent elution yields suspensions containing up to 95% infected erythrocytes. Preliminary immunoblotting studies indicate a parasite antigen of 35,000 mol. wt might be implicated in the binding.     

High temperature conjugation of proteins with carbohydrates

A new procedure was used to conjugate lactose and dextran with BSA without using coupling or activating reagents. The method is simple, rapid and cheap. Reducing sugars covalently bind to proteins when lyophilized together and briefly heated to a high temperature.     

Characterization of collagenous meshworks by volume exclusion of dextrans.

The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754--1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)–dextran derivative conjugate

Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble–insoluble poly(N-isopropylacrylamide)–dextran derivative conjugate, named poly(NIPAAm)–DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)–DD conjugation and characterization. On the other hand, the poly(NIPAAm)–DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)–DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Human factor XII (Hageman factor) autoactivation by dextran sulfate. Circular dichroism, fluorescence, and ultraviolet difference spectroscopic studies.

The first event leading to the activation of the plasma kallikrein-kinin system is the surface-dependent conversion of factor XII to an active enzyme. Factor XII autoactivation was investigated using dextran sulfate as a soluble activating surface, and the significance of aggregation and the nature of the conformational change were examined by ultraviolet difference spectroscopy, fluorescence and circular dichroism. Results indicate that DS500 (500-kDa dextran sulfate) induces aggregation of factor XII. Analysis of the binding data suggests that 165-192 factor XII molecules can bind to one DS500 chain, while a 1:1 stoichiometry is observed with 5-kDa dextran sulfate. The interaction of factor XII and dextran sulfate is a biphasic process. It is initiated by a fast contraction of the molecule upon binding, as revealed by an apparent increase in organized secondary structures, and then followed by a slow relaxation process during cleavage and subsequent activation. Overall, the results are consistent with a model in which factor XII undergoes conformational changes upon binding to the activating surface. The rapidity of autoactivation in the presence of DS500, as opposed to 5-kDa dextran sulfate, implies that aggregation provides a special mechanism whereby proteolytic cleavage is accomplished efficiently when factor XII molecules are bound side by side on the DS500 molecule.