The Binding of Glycoconjugates to Human‐Milk d‐Galactosyltransferase

Abstract

Through the use of affinity chromatography, a homogeneous preparation of human /?( 1 -4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human r-lactalbumin.A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and to /j( 1 + 4)-linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor molecules. Furthermore, experiments with N-acetylglucosamine-/3( 1 + 4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent K,,, values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, a-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides.Although a-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-ghKOSe resulting in the production of lactose, no transfer of galactose was observed to P(1+4)-linked glucose oligomers or to a collagen glycopeptide, D-glucopyranosyl-a( 1 +2)-~-galactopyranosyloxy-P( 1 --f 5)-lysine. It therefore appears that z-lactalbumin can only modify human A protein for monosaccharide acceptors. ability of the human , / I ( 1 -+4)-~-galactosyltransferase. Furthermore, attempts were made to ascertain whether human a-lactalbumin could function in its specifier role [l] with A protein in the presence of a glycopeptide isolated from chick collagen containing a glucosyl residue at the non-reducing end and also with /?( 1 -4)-linked D-glucose oligomers.On the basis of our results, we have been able to arrive at some conclusions concerning the number of sugar binding subsites of human A protein. EXPERIMENTAL PROCEDURE MuteriulsOvalbumin (type V), peptidase, protease, trypsin, chymotrypsin, UDP-D-galactose and GlcNAc were purchased from Sigma Chemical Co. UDP-D-['4C]-galactose (467 counts min-' nmol-') was from the Radiochemical Centre, Amersham. The cell wall glyco-

Design of Dual‐Emission Chemosensors for Ratiometric Detection of ATP Derivatives

“…[18] www.chemasianj.org sugars are converted into nucleoside PPs during glycosyl transfer, detection of the nucleoside PPs by our chemosensor enables us to monitor this reaction without perturbation of the reactants (Scheme 2). [21] The chemosensor-based glycosyltransferase assay presented herein is precise and convenient, and does not require any special modification of either glycosyl donor or acceptor. Thus, we monitored by fluorescence the glycosyl transfer from UDP-Gal to N-acetyl glucosamine (GlcNAc) catalyzed by b-1,4-galactosyltranferase in the presence of 2-2Zn II (Scheme 2).…”

“…The change in the emission ratio was converted into the amount of UDP by a calibration curve, and the data, analyzed with a Lineweaver-Burk plot, gave the Michaelis constant K M as 1.1 mm (Figure 8 b), a value of the same order as that reported (5.1 mm). [21] The chemosensor-based glycosyltransferase assay presented herein is precise and convenient, and does not require any special modification of either glycosyl donor or acceptor. Therefore, this should provide a potentially general method for probing glycosyltransferase activities, which may be superior to conventional methods with radio-labeling or enzyme-reaction-coupling techniques.…”

Biochemical investigations of retinotectal adhesive specificity

“…Other transferases that have been examined have been shown to be specific for both the sugar they transfer and for the acceptor to which the transfer occurs (31). The latter recognition implies a molecular complementarity between transferase and acceptor and has prompted the hypothesis by Roseman (32) that such enzyme-substrate interac- Fig.…”

endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins.

“…2) galactose by galactosyltransferase. This enzyme has previously been shown to transfer galactose to nonreducing N-acetylglucosamine residues ofglycoproteins (19). Thus, ifendo F cleaved the N,N'-diacetylchitobiose core (like endo H and endo D), a nonreducing N-acetylglucosamine would be exposed and galactose transfer would occur.…”