Galactosyltransferase, reactions

Finally, UDP-galactose is epimerized to UDP-glucose by the action of UDP-galactose epimerase (see Figure 12-6). This UDP-glucose can be used in the galactosyltransferase reaction. 

In our prior experience, employing aminoethyl-substituted poly(acrylamide) gel beads as acceptors in the galactosyltransferase reaction, the transfer yields were lower than 1% compared to a 30% yield in the case of a soluble acceptor-polymer (5). Longer spacer arms were tried, with particular attention to the swelling of the resulting polymers, and the... 

Figure 4. Galactosyltransferase reaction with a solution polymeric acceptor followed by photochemical release of the product from the polymer.

Scheme 5. Proposed transition-state structure for the pi,4-galactosyltransferase reaction.

Most of these reactions take place in the cystosol of the epithelial cells surrounding the alveoli of the mammary gland, but the final step, in which the galactosyltransferase is modified by a-lactalbumin, occurs... 

The novel three-enzyme-reaction cycle was further combined with recombinant al-3 galactosyltransferase to synthesize the important trisaccharide... 

Galactosyltransferase (0.05-0.25 mU) was assayed in 50 /u.L of 50 mM cacodylate-HCl buffer (pH 7.4) containing 0.4 mM UDP-galactose, 25 mM glucose, 30 mM MnCl2,0.2 mg/mL of a-lactalbumin, 1.0 mg/mL bovine serum albumin, and 0.1 mM a-L-fucose (internal standard). The reaction was stopped by the addition of 1 mL of cold distilled water. Samples were processed as described above. The mobile phase for HPLC was 100 mM NaOH, and a detector setting of 0 to 3.0 fiA was used. 

Fig. 2. Reactions catalyzed by galactosyltransferase (GT). (a) The incorporation of galactose (Gal) into a (1—>4) linkage with JV-acelylglucosamine (NAG) to form N-acetyl-lactosamine (NAL). UDP, Uridine diphosphate, (b) Modification of the activity of GT by a-lactalbumin (a-LA) to convert it to a lactose synthase catalyzing the formation of lactose from UDP-Gal and glucose.

Powell and Brew (1974a) demonstrated the presence of galactosyltransferase and glycosyltransferase in the Golgi membrane of onion stem. The former showed many similarities to the animal enzyme. It was manganese dependent and gave the same reaction (apparendy) as the animal enzyme. It had a similar to that of A -acetylglucosamine (5.2 mM) and could be activated by bovine a-lactalbumin. 

These findings help to differentiate between possible ways in which a-lactalbumin could influence the production of lactose. One of these, which, on the surface, appears plausible, is that a-lactalbumin might accept iV-acetyllactosamine as a substrate, this product having arisen from the enzymatic action of galactosyltransferase. The ensuing transglycosyl-ation, whereby lactose would be produced, could then account for the effect of a-lactalbumin in the presence of galactosyltransferase. Brew et al. (1968) showed, however, that a-lactalbumin has no affinity for A -acetyllactosamine and thus could not he involved in this reaction. 

An alternative possibility involves an induced conformational change in a-lactalhumin as a result of complexing with galactosyltransferase, so as to produce an affinity for A -acetyllactosamine. Although this explanation appears never to have heen ruled out, there would be no need to invoke it, since it is already known (Klee and Klee, 1970, 1972 Andrews, 1969 Fitzgerald et al., 1970a) that galactosyltransferase has the innate ability to carry out this reaction, even in the total absence of a-lactalbumin. 

Khatra et al. (1974) studied, by steady-state kinetics, the reactions catalyzed by human milk galactosyltransferase. Whether in the presence or absence of a-lactalbumin, they concluded that the reactants added in the order Mn(II), UDP-galactose, and monosaccharide. They felt, however, that their kinetic results could best be explained with a mechanism whereby a-lactalbumin attaches to the enzyme immediately before the monosaccharide, contrary to the finding by Morrison and Ebner (1971b). 

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