Hetero-oligosaccharides play important roles in cellular recognition processes, transformation and biological structures (Varki, 1993). Although many of the glycosyltransferases that catalyze the synthesis of different linkages in glycoconjugates have been cloned and sequenced, there is limited information regarding their structures and mechanisms of action. UDP-galactose [beta]-N-acetylglucosaminide [beta]4-galactosyltransferase-1 ([beta]4GT-1; EC 2.4.1.38), a trans-Golgi membrane enzyme that catalyzes the attachment of [beta] galactose to the 4-position of [beta]-linked N-acetylglucosamine, is one of the most extensively characterized glycosyltransferases. Its specificity is not as strict as those of many other glycosyltransferases and it has consequently proved to be useful for the enzymatic synthesis of various natural and unnatural oligosaccharides (Palcic and Hindsgaul, 1991; Ichikawa et al., 1992; Öhrlein et al., 1992). In vivo, [beta]4GT-1 has a well-defined second function as the catalytic component of lactose synthase, the enzyme system that catalyzes and regulates the synthesis of lactose in the lactating mammary gland (Brew et al., 1968; Hill and Brew, 1975). The interaction of [beta]4GT-1 with the lactose synthase regulatory protein, [alpha]-lactalbumin (LA), results in a change in specificity for acceptor substrates so that glucose, a marginal acceptor substrate, is bound with an affinity that is increased by three orders of magnitude. The combination of the two proteins can consequently catalyze lactose synthesis efficiently. LA also promotes the binding of a range of other acceptors, including noncarbohydrate molecules to [beta]4GT-1, further enhancing its synthetic capabilities (Schanbacher et al., 1970; Yu et al., 1995). A fraction of [beta]4GT-1 is found on the surfaces of some cells which may act as an adhesion molecule by binding to oligosaccharides in the extracellular matrix and on the surfaces of other cells (Shur, 1991); in this context, roles have been attributed to [beta]4GT-1 in a wide range of cell-cell interactions (Shur, 1983; Bayna et al., 1988; Miller et al., 1992; Pratt et al., 1993).
The sequences of [beta]4GTs from different sources indicate, as for other glycosyltransferases (Appert et al., 1986; Narimatsu et al., 1986; Shaper et al., 1986, 1988), the presence of a short N-terminal cytosolic region, a helical hydrophobic transmembrane segment, a stem of about 60 residues and a C-terminal catalytic region (Paulson and Colley, 1989). Recently, five additional homologous [beta]4GTs have been identified in human ([beta]4GT -2 to -6) which differ in acceptor substrate specificity and sensitivity to [alpha]-lactalbumin (Almeida et al., 1997; Lo et al., 1998; Schwientek et al., 1998). Four [beta]4GT-1 homologues have been identified in invertebrates, a UDP-GlcNAc : [beta]-GlcNAc GlcNAc transferase from the pond snail (Bakker et al.,1994), a protein from C.elegans that has a similar domain structure to [beta]4GT-1, a second additional C.elegans clone that encodes a molecule that lacks the stem region and the N-terminal section of the catalytic domain; we have also recently identified a homologous EST from Drosophila (accession number AA142310 from the dbEST database). When all [beta]4GT homologues are compared, a few highly conserved regions of sequence are found that include a region corresponding to residues 305-324 of bovine [beta]4GT-1 [NGfpNnYwGWGgEDDdiynR] (Figure
Figure 1. Alignment of the region conserved in (A) [beta]4GT (Shaper et al., 1997; Lo et al., 1998), (B) Lex1, (C) FNG, (D) BRN, and (E) [beta]1,3 galactosyltransferase subfamilies. Residues conserved within subfamilies are boldfaced. The conserved patches among all the sequences are shaded. Angled brackets indicate the omission of amino acids flanking the conserved sequences.
Truncated forms of [beta]4GT-1 have been previously expressed in yeast (Krezdorn et al., 1993; Herrmann et al., 1995) and E.coli (Boeggeman et al., 1993; Nakazawa et al., 1993). With the yeast system, using fermentation technology, yields of secreted [beta]4GT-1 are about 700 mU/l (~0.3 mg). [beta]4GT-1 has also been expressed in E.coli as inclusion bodies, from which folded protein has been generated in yields of about 1 mg/ liter (Boeggeman et al., 1993); similar low yields have also been obtained using an E.coli secretion vector (Nakazawa et al., 1993). With these systems insufficient protein has been obtained for characterizing the effects of mutations on structure as well as activity. We describe here the expression of bovine [beta]4GT-1 using the pET15b vector, with which a truncated form of the catalytic domain is produced with a N-terminal 6-histidine tag, allowing the protein extracted from inclusion bodies with denaturants to be readily purified under denaturing conditions. Conditions for folding the protein in high yields have been devised, to provide up to 100 U/l of bacterial culture. After purification the recombinant enzyme has kinetic and regulatory properties as well as a CD spectrum that are closely similar to the wild-type enzyme. Mutagenesis studies with this system indicate that the conserved cluster of anionic residues is crucial for the catalysis and not for the binding of the metal cofactor or substrate; a possible role in transition state stabilization is discussed.
Expression of [beta]4GT
[beta]4GT-1 was expressed as a His-Tag fusion protein using the pET15b vector to facilitate purification under denaturing conditions prior to folding. pET3a is an efficient vector for expressing recombinant truncated [beta]4GT-1 (designed as r[beta]4GT-1) as inclusion bodies from which the protein can be extracted with urea and other denaturants, but only low levels of folding (~2%) were achieved using a variety of procedures and large amounts of precipitate and low yields of active enzyme were obtained when folding was attempted by dialysis or dilution methods. This appears to result from the interaction of the highly cationic [beta]4GT-1 molecule (pI 9.6) and negatively charged nonprotein components, such as nucleic acids that are also present in inclusion bodies, since the truncated [beta]4GT-1 extracted from inclusion bodies with 8 M urea did not bind to cation exchange columns at pH 6 (Malinovskii and Brew, unpublished observations). However, protein extracted with 6 M guanidine HCl from inclusion bodies produced by expression from the pET15b vector was readily purified by Ni2+ chelate chromatography. Using the protein purified in this manner, 70-100 U of active enzyme were generated from the protein expressed in a liter of bacterial culture; this corresponds to 10-15 mg of folded r[beta]4GT-1 (Table II and Table III). The presence of a redox system is necessary for generating active enzyme, indicating the need for disulfide bond formation during folding.
Table I.
Primer | Sequence | Orientation |
XhoI-GT | CCTTTGTATGTGCAATTCG | Complementary |
StuI-GT | CAGTTAGACTATGGCATC | Coding |
D318N:D320N | TGGGGAGGTGAAAACGATAACATTTATAACAGA | Coding |
D318N | TGGGGAGGTGAAAACGATGACATTTATAACAGA | Coding |
D318E | TGGGGAGGTGAAGAGGATGACATTTATAACAGA | Coding |
W312F | TTTCCTAATAACTACTTCGGCTGGGGA | Coding |
Table II.
Protein | Yield (mg/l of culture) |
r[beta]4GT-1 | 10.0 |
D318N:D320N | 5.3 |
D318N | 6.1 |
D318E | 7.0 |
W312F | 11.0 |
Properties of recombinant [beta]4GT-1
The folded enzyme, isolated after chromatography in the absence of denaturant showed a single band on SDS-polyacrylamide gel electrophoresis with a mobility close to the 31 kDa marker, which is consistent with the predicted molecular weight of 33 kDa (Figure
Figure 2. SDS-PAGE analysis of wild type recombinant [beta]4GT-1 or mutants D318E, D318N, W312F, and D318N:D320N after folding; 1.5 µg/lane of sample in 20 mM Tris-HCl pH7.4 and 50% glycerol were loaded to the gel with sample loading buffer containing 0.36 M Tris-HCl, pH6.7/10% SDS/40% glycerol/50 mM DTT/0.005% bromophenol blue. Lanes 1-5 are wild type [beta]4GT-1, D318E, D318N, D318N:D320N, and W312F, respectively.
Figure 3. Far (A) and near (B) UV circular dichroism spectra of r[beta]4GT-1. These were determined at a protein concentration of 0.23mg/ml at 25°C. [solid line] Wild type; [-·-·-·-] W312F; [··-··-··] D318E; [········] D318N:D320N; [· · · · ·] D318N. Differences between the spectra can be accounted by the effects of the substitution itself on the CD spectrum, as opposed to structural perturbations. Near UV CD spectra have multiple peaks and troughs resulting from the fixed asymmetric environments of aromatic side chains and disulfide bonds while far UV CD spectra are strongly influenced by secondary structure (Woody, 1995). Differences between the single site mutants are small and appear to be effects of the substitutions on the environments of aromatic residues or, in the case of the Trp312 to Phe mutant, the direct contribution of the side chain of this residue to the spectrum. This mutant shows the largest change in the far UV region that is likely to reflect the contribution of Trp312 to the spectrum in this region. The near UV CD spectrum of the double mutant is weaker but similar in shape to those of the other proteins and is minor compared to changes observed in structurally perturbed mutants of other proteins (e.g., see Huang et al, 1997).
Analysis of steady state kinetic data at a fixed concentration of Mn2+ in which the concentrations of both UDP-galactose and GlcNAc substrates are varied or the concentrations of LA and glucose are varied at a fixed concentration of UDP-galactose gives values for kinetic parameters (Table III) that are similar to those previously reported for bovine [beta]4GT-1 (Powell and Brew, 1974) except the specific activity (the kcat) for r[beta]4GT-1 is 3- to 4-fold lower than that of the enzyme purified from milk.
Mutants of r[beta]4GT-1
As discussed previously, mutations were introduced into r[beta]4GT-1 to investigate the possible role of a conserved region of sequence W312GWGGEDDD320, which represents a plausible location for a binding site for the catalytically essential cation. Because of the highly conserved nature of this region, substitutions were designed to be structurally conservative and to introduce changes in size or charge; the goal was to perturb but not eliminate activity so that the roles of residues can be examined quantitatively through their effects on kinetic parameters. Phenylalanine was substituted Tryptophan 312 since this substitution is present in the homologous UDP-GlcNAc : [beta]-GlcNAc GlcNAc transferase from the pond snail (Bakker et al.,1994). Aspartates 318 and 320 were selected for mutagenesis since they form a DXD motif that is found in many glycosyltransferases and hydrolases (Breton et al., 1998). Substitutions were made of asparagine and glutamate for aspartate 318, and asparagine for both aspartates 318 and 320 using the mutagenic primers listed in Table I.
The entire sequences of the mutants were checked by DNA sequencing, confirming the presence of the desired mutation in each case and that no unwanted mutations were introduced by the PCR mutagenesis procedure. All proteins expressed at similar levels as inclusion bodies and behaved similarly during the folding process; folded forms of the proteins were isolated in final yields of 6-11 mg/l of culture after the chromatography and ammonium sulfate precipitation steps (Table II) and were homogeneous on SDS-gel electrophoresis (Figure
Figure
Table III.
Ligand | Parameter | Bovine [beta]4GT-1 | r[beta]4GT-1 | D318N | D318E | D318N: D320N | W312F |
UDP-galactose | Kia | 0 | 0 | 0.12 ± 0.02 | 1.7 ± 0.5 | NAa | 0.15 ± 0.04 |
Ka | 0.28 mM | 0.13 ± 0.02 mM | 0.10 ± 0.03 mM | 0.7 ± 0.2 mM | 0. 9 ± 0.2 mM | ||
GlcNAc | Kb | 25 mM | 14 ± 2 mM | 21 ± 3 mM | 18 ± 5 mM | NAa | 64 ± 9 mM |
Bovine LA | Kc | 0 | 0 | NAa | NAa | NAa | 0 |
Kic | 25.5 µM | 27 ± 4 µM | 36 ± 8 µM | ||||
Mn2+ b | K1 | 20 µM | 37 ± 8 µM | 17 ± 1 µM | 31 ± 21 µM | NAa | 17 ± 6 µM |
K2 | 1 mM | 0.92 ± 0.06 mM | 0.55 ± 0.09 mM | 1.9 ± 0.7 mM | 2.0 ±0.1 mM | ||
glucose | Kd | 1.7 mM | 2.7 ± 0.5 mM | NAa | NAa | NAa | 0.43 ± 0.09 mM |
Spec. activity | 26 (µmol/min/mg) | 7.7 ± 0.6 (µmol/min/mg) | 0.049 ± 0.005 (µmol/min/mg) | 0.051 ± 0.005 (µmol/min/mg) | 0.003 (µmol/min/mg) | 8.1 ± 0.7 (µmol/min/mg) |
Functional properties of mutants of r[beta]4GT-1
[beta]4GT-1 mutants with substitutions for Asp318 and 320 were much less active than the wild-type protein and were assayed at higher protein concentrations. Mutant enzymes were analyzed with respect to Mn2+-activation in comparison with the wild-type protein. Data fitted to an equation describing two metal-binding sites (Powell and Brew, 1976) indicate insignificant differences between the mutants and wild-type protein (Table III). Other kinetic analyses were conducted at an essentially saturating concentration of metal ion (10 mM). Initial velocity data obtained with varying concentrations of UDP-galactose at a series of fixed concentrations of GlcNAc give essentially parallel lines in double reciprocal plots for the wild-type protein and fit best to a rate equation that lacks a Kia term (Equation 2). The parameters for these mutants are summarized in Table III. Compared to wild type r[beta]4GT-1, the two mutants with substitutions for Asp318, asparagine, and glutamic acid have only 0.1% of the activity of the wild-type protein, and the double mutant, D318N:D320N had ~0.01% activity of wild type r[beta]4GT-1. In contrast, the Trp312 to Phe mutant has a similar activity to the wild type protein. Steady state kinetic parameters determined by a detailed analysis of the single-site mutants are summarized in Table III; however, the extremely low level of activity of the double mutant precluded this type of analysis. The results indicate that the similar kinetic parameters associated with Mn2+, GlcNAc and UDP-Gal, are similar to those of the wild-type protein except in the case of D318E, Kia, the equilibrium dissociation constant for UDP-Gal binding from the enzyme·Mn2+ complex was much greater. This change is illustrated in Figure
Figure 4. Comparison of steady state kinetic properties between mutants (A) D318N and (B) D318E. The double reciprocal plots show variation of N-acetyl-lactosamine synthase activity with the concentrations of UDP-galactose and GlcNAc at 10 mM Mn2+. UDP-galactose is plotted as the variable substrate at the following fixed concentrations of GlcNAc. (A) solid circles, 6 mM; open circles, 7.5 mM; inverted solid triangles, 10 mM; inverted open triangles, 15 mM; solid squares, 30 mM. (B) solid circles, 6 mM; open circles, 10 mM; inverted solid triangles, 15 mM; inverted open triangles, 30 mM; solid squares, 50 mM.
Previous mutational studies of soluble truncated forms of [beta]4GT-1 have utilized low yield bacterial secretion expression systems (Aoki et al., 1990; Zu et al., 1995). Substitutions of Gly for Tyr286, Tyr311, or Trp312 (sequence numbering is given in Figure
Our results show that conservative substitutions for Asp318 and 320 do not alter the ability of r[beta]4GT-1 to fold to a correct native structure and produce relatively small changes in the binding of the Mn2+ cofactor and acceptor and donor substrates. However, there is a large reduction in kcat with a magnitude comparable to that obtained when residues of the catalytic triad are mutated in serine proteases (Fersht and Sperling, 1973). kcat/(Kia*Kb), a parameter for bisubstrate reaction that is related to transition state stabilization and is a measure of catalytic efficiency, for D318N and D318E is decreased by 3 and 5 orders, respectively, compared to that of human milk [beta]4GT-1 (Khatra et al., 1974). The substitution of Phe for W312, six amino acids away from the highly conserved DDD (318-320) patch, did not perturb the enzyme properties. These results suggest that these highly conserved aspartates in [beta]4GT-1 have a critical role in catalysis.
The kinetic mechanism of [beta]4GT-1 is sequential, in keeping with a displacement mechanism through which the 4-OH of the acceptor displaces the phosphoryl-galactose bond in the donor substrate, resulting in the inversion of configuration at the galactose C1. Secondary deuterium isotope effects observed with [1-2H]-UDP-galactose (Kim et al., 1988) and competitive inhibition by UDP-(2-deoxy-2-fluoro)-galactose (Hayashi et al., 1997) indicate that the galactose transfer reaction mechanism involves an intermediate in which the anomeric C of galactose has sp2 character and the UDP to galactose bond is substantially cleaved in the transition state. The stabilization of a cationic galactose moiety by this anionic region of [beta]4GT-1 in the transition state provides a plausible, but not unique, explanation of the results reported here. Aspartates 318 and 320 do not directly affect substrate and metal ion binding, but do affect the interaction between the enzyme and the transition state in the catalytic mechanism.
Previously, a conserved DXD motif has been noted in many prokaryotic and eukaryotic galactosyltransferases (Breton et al., 1998) and other glycosyltransferases (Wiggins and Munro, 1998). It has been speculated that these residues may constitute a binding site for the divalent metal ion that is required for catalysis by many of these enzymes (Wiggins and Munro, 1998). [beta]4GT-1 and its homologues also have a conserved DVD motif that is located about 60 residues N-terminal to the acidic cluster investigated here. Our results show that the EDDD sequence of [beta]4GT-1 is not the binding site for the essential metal cofactor and suggest that it is possible that not all of the conserved acidic clusters in the various glycosyltransferases are functionally and structurally equivalent. The region investigated here appears more similar to one that is highly conserved among the FNG, BRN, Lex1 families (Yuan et al., 1997) and [beta]1,3 galactosyltransferase subfamilies (Amado et al., 1998; Kolbinger et al., 1998) (Figure Materials
pET3a and pET15b expression vectors and T7 promoter and T7 terminator primers were obtained from Novagen, Madison, WI. Restriction enzymes and DNA ligase were supplied by New England BioLabs, Beverly, MA. UDP-[3H]-galactose was from NEN Products, Boston, MA. Guanidine hydrochloride (Ultrapure) was purchased from J.T. Baker, Inc., Jackson, Tenn. His-Bind resin was from Novagen. Expression
The region of the cDNA for bovine [beta]4GT-1 encoding residues 129-402 of the protein sequence was amplified by PCR using the synthetic primers:
Discussion
Materials and methods
5[prime] GTG CCC TCC ACC CAT ATG CGC TCG CTG ACC GCA3[prime]
Met Arg Ser Leu Thr Ala
2. (C-terminal; complementary)
5° GAT CAG TGC ACC GGA TCC CTA GCT GCT CGG CGT CCC3[prime] (stop)
PCR reactions were performed using a Perkin Elmer/Cetus thermocycler for 25 cycles. The product was gel purified, digested with NdeI and BamHI and cloned into preparations of the pET15b vector that had been previously treated with the same enzymes. The product was transformed into E.coli.DH5[alpha] competent cells. The selected clones were grown and used for plasmid minipreps. The purified plasmid construct, designed as pET15b-r[beta]4GT-1, was used for DNA sequencing to confirm that no mutations were introduced by PCR. The pET15b-r[beta]4GT-1 was used to transform BL21(DE3) cells and the recombinant [beta]4GT-1 was expressed using previously described methods (Grobler et al., 1994). Cells were harvested and lysed using lysozyme and deoxycholate. Inclusion bodies were collected by centrifugation and washed.
Purification and Folding of r[beta]4GT-1
Inclusion bodies were dissolved in 6 M guanidine hydrochloride containing 20 mM Tris-HCl pH 7.9, 0.5 M NaCl, and 5 mM imidazole. The extract is applied to a column containing 10 ml Ni2+-charged His-Bind resin. After washing, with 20 mM imidazole, protein is eluted with 200 mM imidazole. All buffers contain 6 M guanidine hydrochloride, 0.5 M NaCl, and 20 mM Tris-HCl pH 7.9.
To determine the protein concentration, the absorbance of enzyme solutions were measured at 280 nm and the concentration was determined using a value for E280nm0.1% of 1.23 calculated from the amino acid composition (Mach et al., 1992).
For folding, the solution containing purified r[beta]4GT-1 is diluted to a concentration of 0.1mg/ml with the same buffer. Dialysis is carried out at 4°C in a Macro DiaCell system (InstruMed Inc., Union Bridge, MD) fitted with 6-8000 molecular weight cutoff membranes against 5 volumes of Tris-HCl buffer pH 7.5 containing 20 mM imidazole, 10% glycerol, 10 mM [beta]-mercaptoethanol, and 1 mM 2-hydroxyethyl disulfide. The dialysis solution is changed four times at 18-24 h intervals. Up to 400 ml of r[beta]4GT-1 solution can be accommodated in one apparatus producing, reproducibly about 50 units. Folded r[beta]4GT-1 is precipitated between 10 and 80% ammonium sulfate and stored at -20° in 50% glycerol. Further purification can be carried out by affinity chromatography with LA-Sepharose in the presence of N-acetyl-glucosamine (Trayer and Hill, 1971).
Mutagenesis of [beta]4GT-1
Mutations were introduced by PCR using the "megaprimer" method (Sarker and Sommer, 1990) with pET15b-r[beta]4GT-1 construct as the template. The amplification to generate the megaprimer was performed with the synthetic StuI primer or XhoI primer together with an appropriate mutagenic primer. Table I lists the StuI and XhoI primers together with the mutagenic primers that were used for the different substitutions. The megaprimer was purified by agarose gel electrophoresis and used in a second amplification with the same template and the cognate StuI or XhoI primer. After purification by agarose gel electrophoresis and a Promega PCR purification kit, the final amplified product was digested with StuI and XhoI and cloned into the pET15b-r[beta]4GT-1 vector which was previously made by cleaving pET15b-r[beta]4GT-1 construct with the same enzymes. The product was transformed into E.coli DH5[alpha] competent cells, and selected clones were grown and used for minipreps to provide plasmid for characterization by restriction mapping and DNA sequencing.
Enzyme assays
Galactosyltransferase assays with GlcNAc and glucose (in the presence of LA) were performed by a radiochemical method as described previously (Brew et al., 1968). r[beta]4GT-1 and the Trp312Phe mutant were assayed at a final concentration of 4.6 µg/ml and other mutants at concentrations of 92 µg/ml. For characterization of metal-dependence, Mn2+ was added in a concentration range of 2 µM to 20 mM in the presence of 0.3 mM UDP-galactose and 10 mM GlcNAc.
The data were fitted to a single substrate Michaelis-Menten equation and also to an equation describing metal binding to a high affinity site (K1) to generate an enzyme form with a low kcat (V1) and to a second lower affinity site (K2) to generate a form with a higher kcat (V2):
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Kinetic parameters for UDP-galactose and GlcNAc were determined using concentrations varying from 0.1-2.0 mM and 6-50 mM, respectively, at 10 mM MnCl2. Kinetic parameters associated with the action of LA using LA in a concentration range of 0-40 µM as an inhibitor of galactose transfer to N, N[prime]-diacetylchitobiose to determine the dissociation constant (Ki) of LA from the Enzyme·Mn2+·UDP-galactose·LA complex (Grobler et al., 1994; Malinovskii et al., 1996). The promotion of lactose synthase at a fixed concentration of glucose (10 mM) by 0.5-3.6 µM LA and the data were deconvoluted as described previously (Grobler et al., 1994; Malinovskii et al., 1996) to determine the Km for glucose at a saturating concentration of LA.
Kinetic data were fitted to appropriate rate equations using the Curvefitter algorithm of SigmaPlot (Jandel Corp.). The following rate equations were used for different sets of data.
1. General equation for sequential symmetrical initial velocity pattern (ordered or random equilibrium sequential mechanism):
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2. Equation for an asymmetric initial velocity pattern associated with a ping pong mechanism or sequential mechanism in which substrate A does not dissociate well from the E·S complex:
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CD spectroscopy
Near and far UV CD spectra of recombinant proteins were determined with a JASCO J-710/720 spectropolarimeter. Twenty spectra were scanned for each sample at a speed of 100 nm/min which were subsequently averaged and smoothed. Near UV CD spectra (250-320 nm) were determined using a cell with a path length of 1 cm, and far UV spectra (200-250 nm) using a cell with a path length of 0.1 cm. Proteins were dissolved in 20 mM Tris-HCl, pH 7.4, containing 50% glycerol at concentration between 0.15 and 0.5 mg/ml.
Other methods
Oligonucleotide synthesis and DNA sequencing was carried out by Dr. Rudolf Werner, Department of Biochemistry and Molecular Biology, University of Miami.
We thank Dr. Joel H.Shaper, Department of Oncology, John Hopkins University, School of Medicine for providing the cDNA for bovine [beta]1,4 galactosyltransferase-1. This work was partially supported by grant GM21363 from NIH. This work was supported in part by Research Grant GM21363 from the National Institutes of Health. The costs of publication were defrayed in part by the payment of page charges. This article must thereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
[beta]4GT-1, UDP-galactose [beta]-N-acetylglucosaminide [beta] 1,4 galactosyltransferase; LA, [alpha]-lactalbumin; CD, circular dichroism.
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