Jean-Paul Prieels

[41] Purification of mammalian glycosyltransferases

Publisher Summary It is reported that purified glycosyltransferases are powerful tools for the study of glycoconjugate structure and function, as well as the regulation of glycoprotein biosynthesis. Thus, this chapter presents the procedure for purification of seven mammalian glycosyltransferases. Affinity chromatographic methods are well suited to the isolation of glycosyltransferases. The technique tends to separate enzymically active species from inert related forms. Because there may be great specificity in both the adsorption and elution steps, the extraction of minor constituents from complex protein solutions is often feasible. The conditions for optimal adsorption are frequently similar to those for maximum enzymic activity, which for most enzymes is close to conditions for optimum stability. The addition of detergents, if necessary, does not generally interfere with the mechanism of affinity chromatography. Most methods of elution give a concentrated enzyme solution that may enhance the stability of labile proteins that tend to denature if diluted. Finally, knowledge of kinetic parameters permits a rational approach to the choice of an affinity ligand, as well as conditions for adsorption and elution.

Lactofferrin binding to lysozyme-treated Micrococcus luteus

When the cell lysis of Micrococcus luteus by hen egg white or human lysozyme is performed in the presence of bovine or human lactoferrin, a temporary increase of the turbidity of the solution as followed at 450 nm is observed. Examination of the suspension under light microscopy has proven that the protoplasts produced upon lysozyme action are agglutinated by lactoferrin. The rate of agglutination depends on pH, lactoferrin, lysozyme and cells concentrations. Agglutination is maximal at pH 5.5. Around 1.4 X 10(6) binding sites for lactoferrin per cell have been determined through a Scatchard plot analysis. The binding to the cells is not mediated by the glycosidic moiety of lactoferrin but rather by a charge-to charge interaction as succinylation of about four out of the 39 lysines of lactoferrin completely abolishes its ability to agglutinate the cells. Binding does not depend on ionic iron nor on the iron content of lactoferrin itself.

Reversible inactivation of lactose synthase by the modification of his 32 in human α-lactalbumin

Summary Treatment of human α-lactalbumin with diethylpyrocarbonate at pH 6.1 resulted in reduction of its activity in the lactose synthase assay. Almost complete loss of activity occurred when the molar ratio of reagent to protein was 15:1. Incubation of the modified protein in 20 mM hydroxylamine for 1 h restored its full activity. The loss of activity was correlated with the ethoxyformylation of one of the two histidine residues of human α-lactalbumin, as measured by the change in absorption at 242 nm. Evidence is provided that histidine is the only amino acid residue modified. Examination of the NMR spectrum of the inactivated protein demonstrated the disappearance of one histidine (C-2 proton) peak, which could be assigned to that of His 32.

Characterization of Two Highly Purified Fucosyltransferases

Publisher Summary This chapter describes the characters of the two highly purified fucosyltransferases. Fucosyltransferases catalyzes the transfer of L-fucose from GDP–Fuc to carbohydrate acceptors by linking α-(1→2) to Gal, α-(1→4) to GlcNAc, α-(1→3) to GlcNAc and Glc, and α-(1→6) to GlcNAc.Two of these enzymes, a GDP-Fuc:β-D-Gal 2-α-L-fucosayltransferase [α(l→2)FucT] from porcine submaxillary glands, and a GDP–Fuc : (β-D-Gal 3-α-L-fucosyltransferase [α(l→3)FucT] from human milk, are purified and partially characterized. This represents a124,000-fold purification in an overall yield of 6%. Comparison of the apparent Km values for acceptors indicates a preference for structures having the β-D-Gal-(1→3)-HexNAc linkage. The cells were not agglutinated by η-specific lectins after treatment with the α(l→2) FucT alone but were agglutinated if pretreated with neuraminidase. However, it was also found that fucose were incorporated at approximately106 residues per cell when treated with the α(l→2) FucT alone and that these cells could be agglutinated with the Hspecific lectins. This suggests that sialic acid does not block the incorporation of fucose but rather interferes with the binding of the lectin to the cells.

The influence of membrane mutations on metastasis.

In an effort to assess the effect of surface carbohydrates upon the metastasizing properties of tumor cells, lectin-resistant mouse melanoma cells were selected. Wheat-germ-agglutinin-resistant lines displayed mainly decreased metastasis properties as well as well-defined alterations in surface carbohydrates: in a glycopeptide with four side chains, two of them were missing their terminal sialic acid residues while two fucoses were newly attached to the oligosaccharide. The enzymatic defect could be pinpointed to an over-60-fold increase in fucosyltransferase, while the sialyltransferase did not decrease significantly. Revertants were again selected with lectins and their fucosyltransferase activities returned to normal values again. The metastasizing potential of the revertants was not yet assessed carefully but a return of some of the metastasizing potential was noted.

A fluorimetric study of the interactions of insolubilized human α-lactalbumin with galactosyl transferase (A-protein) and with anti-α-lactalbumin antibodies

Abstract Intrinsic as well as extrinsic fluorescence of an immobilized protein was used for the study of the interactions between α-lactalbumin-Sepharose and protein ligands. The fluorescence peak of the human α-lactalbumin-agarose conjugate was shifted to the blue and quenched in the presence of the galactosyl transferase (A-protein), indicating the probable formation of a complex between both proteins. The natural fluorescence of human α-lactalbumin bound to Sepharose was specifically quenched in presence of antihuman α-lactalbumin antibodies. This change in fluorescence appears to be due to binding of the antibodies to the immobilized antigen. Furthermore, the extrinsic fluorescence of a bound dye such as 2- p -toluidinylnaphthalene-6-sulfonate was used to confirm the existence of binding between antibodies and α-lactalbumin-agarose, and to obtain values for the association constant. A value of 5.6·10 +6 M −1 for the binding constant was reported, which compares favorably with other data obtained by equilibrium dialysis.

Disulfide content of reduced hen egg white and human milk lysozymes during the folding process

In order to obtain a better understanding of the possible influence of the primary sequence of a protein on its folding pathway, renaturation of reduced human milk lysozyme was compared to that of reduced hen egg white lysozyme. Following disulfide bond formation, under identical conditions, similar products were found during the folding of both lysozymes, but the kinetics of appearance and disappearance of these intermediates as well as the appearance of the native conformation were different.