GLUT1 and GLUT8 support lactose synthesis in Golgi of murine mammary epithelial cells.

The mammary gland increases energy requirements during pregnancy and lactation to support epithelial proliferation and milk nutrients synthesis. Lactose, the principal carbohydrate of the milk, is synthetized in the Golgi of mammary epithelial cells by lactose synthase from glucose and UPD galactose. We studied the temporal changes in the expression of GLUT1 and GLUT8 in mammary gland and their association with lactose synthesis and proliferation in BALB/c mice. Six groups were used: virgin, pregnant at 2 and 17 days, lactating at 2 and 10 days, and weaning at 2 days. Temporal expression of GLUT1 and GLUT8 transporters by qPCR, western blot and immunohistochemistry, and its association with lactalbumin, Ki67, and cytokeratin 18 within mammary tissue was studied, along with subcellular localization. GLUT1 and GLUT8 transporters increased their expression during mammary gland progression, reaching 20-fold increasing in GLUT1 mRNA at lactation (p < 0.05) and 2-fold at protein level for GLUT1 and GLUT8 (p < 0.05 and 0.01, respectively). The temporal expression pattern was shared with cytokeratin 18 and Ki67 (p < 0.01). Endogenous GLUT8 partially co-localized with 58 K protein and α-lactalbumin in mammary tissue and with Golgi membrane-associated protein 130 in isolated epithelial cells. The spatial-temporal synchrony between expression of GLUT8/GLUT1 and alveolar cell proliferation, and its localization in cis-Golgi associated to lactose synthase complex, suggest that both transporters are involved in glucose uptake into this organelle, supporting lactose synthesis.

Effects of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in bovine mammary epithelial cells.

As the main precursor for lactose synthesis, large amounts of glucose are required by lactating dairy cows. Milk yield greatly depends on mammary lactose synthesis due to its osmoregulatory property for mammary uptake of water. Thus, glucose availability to the mammary gland could be a potential regulator of milk production. In the present study, the effect of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in vitro was investigated. Bovine mammary epithelial cells (BMEC) were treated for 12 h with various concentrations of glucose (2.5, 5, 10 or 20 mmol/L). The higher concentrations of glucose (10-20 mmol/L) did not affect the mRNA expression of acetyl-CoA carboxylase, diacyl glycerol acyl transferase, glycerol-3 phosphate acyl transferase and α-lactalbumin, whereas fatty acid synthase, sterol regulatory element binding protein-1 and beta-1, 4-galactosyl transferase mRNA expression increased at 10 mmol/L and then decreased at 20 mmol/L. The content of lactose synthase increased with increasing concentration of glucose, with addition of highest value at 20 mmol/L of glucose. Moreover, the increased glucose concentration stimulated the activities of pyruvate kinase and glucose-6-phosphate dehydrogenase, and elevated the energy status of the BMEC. Therefore, it was deduced that after increasing glucose availability, the extra absorbed glucose was partitioned to entering the synthesis of milk fat and lactose by the regulation of the mRNA expression of key genes, promoting glucose metabolism by glycolysis and pentose phosphate pathway as well as energy status. These results indicated that the sufficient availability of glucose in BMEC may promote glucose metabolism, and affect the synthesis of milk composition.

HAMLET: functional properties and therapeutic potential.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.

Lactose synthase components in milk: concentrations of alpha-lactalbumin and beta1,4-galactosyltransferase in milk of cows from several breeds at various stages of lactation.

It is believed that milk production is determined by the number and activity of mammary secretory cells. Secretory activity, as assessed by milk volume, depends on secretion of the major osmole in milk, lactose, which is produced by lactose synthase. The amount of either of the two proteins in lactose synthase may regulate milk production. The objective of this study was to determine whether the concentrations in milk of the two components of lactose synthase, alpha-lactalbumin (alpha-LA) and beta1,4-galactosyltransferase (B4GALT), were related to genetic background, stage of lactation, breed or parity of dairy cows. alpha-Lactalbumin and B4GALT concentrations were measured by ELISA and by enzyme assays, respectively, from single milk samples. Two herds with a total of 279 cows were used in the analysis. One herd contained Ayrshire, Brown Swiss, Holstein and Jersey cows; the second herd contained two groups of cows; Holsteins selected for high milk production and Holsteins with 1960s genetics. The alpha-LA concentration in milk was greater in Jerseys and Ayrshires than in Holsteins and Brown Swiss. However, no difference in alpha-LA concentration was observed in milk from high and low genetic merit cows in the Minnesota herd or among different genetic backgrounds in the Illinois herd. beta1,4-Galactosyltransferase concentrations were similar for all groups that were analyzed. alpha-Lactalbumin concentrations were positively correlated with milk protein concentration, milk fat concentration and lactose concentration. beta1,4-Galactosyltransferase concentration in milk exhibited a strong positive correlation with number of days in milk. Although the concentration of B4GALT increased as lactation progressed, the values did not show any correlation with persistency of lactation or late lactation milk production. In conclusion, this survey shows that the two components of lactose synthase are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.

Structure and catalytic cycle of beta-1,4-galactosyltransferase.

Beta-1,4-galactosyltransferase-1, a housekeeping enzyme that functions in the synthesis of glycoconjugates, has two flexible loops, one short and one long. Upon binding a metal ion and UDP-galactose, the loops change from an open to a closed conformation, repositioning residues to lock the ligands in place. Residues at the N-terminal region of the long loop form the metal-binding site and those at the C-terminal region form a helix, which becomes part of the binding site for the oligosaccharide acceptor; the remaining residues cover the bound sugar-nucleotide. After binding of the oligosaccharide acceptor and transfer of the galactose moiety, the product disaccharide unit is ejected and the enzyme returns to the open conformation, repeating the catalytic cycle.

Beta-1,4-galactosyltransferase and lactose synthase: molecular mechanical devices.

Recent structural investigations on the beta-1,4-galactosyltransferase-1 (Gal-T1) and lactose synthase (LS) have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates (i) the oligosaccharide binding cavity; (ii) a protein-protein interacting site for the enzyme's partner, alpha-lactalbumin (LA); and (iii) a metal ion binding site. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar-nucleotide-enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone, thus making it possible for the protein to act as a specific lectin.

Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I.

The lactose synthase (LS) enzyme is a 1:1 complex of a catalytic component, beta1,4-galactosyltransferse (beta4Gal-T1) and a regulatory component, alpha-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to beta4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to beta4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on beta4Gal-T1, while the N-acetyl group-binding pocket in beta4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for lactose synthase.

Short communication: effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth.

Lactose synthase (a complex of beta1,4-galactosyltransferase and alpha-lactalbumin) forms lactose in the Golgi complex of mammary epithelial cells. To determine whether alpha-lactalbumin is a limiting component in this complex, transgenic mice that expressed bovine alpha-lactalbumin were studied. Transgenic mice produced 0.5 to 1.5 mg/ml of bovine alpha-lactalbumin in their milk, 5- to 15-fold more alpha-lactalbumin than in milk of control mice. Transgenic and control mice produced milk with the same concentrations of lactose, cream, and total solids, and showed similar mammary gland growth, morphology, and histology. Milk from transgenic mice had 0.6% less protein than milk from control mice (P < 0.05). The in vitro lactose synthase activity in mammary gland homogenates from alpha-lactalbumin transgenic mice was increased (P < 0.05), demonstrating that bovine alpha-lactalbumin could interact with murine beta1,4-galactosyltransferase. Pups reared by lactating transgenic mice showed a 4% increase in growth on d 10 of lactation, suggesting that milk production was increased (P = 0.06). Milk volume, estimated using the weigh-suckle-weigh technique, tended to be higher (although not significantly) in transgenic mice (P = 0.11). These results suggest that augmenting alpha-lactalbumin expression in the dam increases the growth of suckling offspring.

Biosynthesis of beta1,4- and beta1,beta1-galactopyranosyl xylopyranosides in the mammary gland of lactating cow.

Lactose is a principal carbohydrate in nearly all species of mammalian milk. In order to examine the acceptor substrate specificity of lactose synthase in vivo, D-xylose as an acceptor substrate was injected into the jugular vein of a Holstein cow during lactation, then a milk sample obtained by milking. beta1, beta1-galactopyranosyl xylopyranoside, a nonreducing disaccharide, was separated from the bovine milk sample after elimination of reducing sugars, identified by fast-atom bombardment (FAB)-MS and 1H-NMR analysis. A mixture of beta1,beta1- and beta1, 4-galactopyranosyl xylopyranoside fractions was also obtained by thin layer chromatography from the milk sample and elucidated by electrospray ionization (ESI)-MS and 1H NMR analysis. Comparison of the integrated intensity of the products shows that the beta1,beta1 and beta1,4 isomers are present in a ratio of 1.0 : 1.4, suggesting that D-xylose, transported from capillary blood across the plasma membrane of the mammary gland, was recognized by lactose synthase in its normal and reverse orientation owing to high symmetry of its structure. While the beta1,4-isomer is known as a fragment of the linkage region between the protein and the polysaccharide chain of proteoglycans, the beta1,beta1-isomer has not been identified in vivo. Here, we demonstrate that galactosylation of D-xylose transported from the capillary blood can occur by lactose synthase catalysis in the mammary gland while the usual galactosylation of D-glucose proceeds. In addition, these results suggest that the possibility of endogenous occurrence of the beta,beta-trehalose type disaccharide in the mammary gland of lactating mammals may not be ruled out.

Expression of functional soluble forms of human beta-1, 4-galactosyltransferase I, alpha-2,6-sialyltransferase, and alpha-1, 3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris.

The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.

Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway.

We have molecularly analyzed three genes, sqv-3, sqv-7, and sqv-8, that are required for wild-type vulval invagination in Caenorhabditis elegans. The predicted SQV-8 protein is similar in sequence to two mammalian beta(1,3)-glucuronyltransferases, one of which adds glucuronic acid to protein-linked galactose-beta(1, 4)-N-acetylglucosamine. SQV-3 is similar to a family of glycosyltransferases that includes vertebrate beta(1, 4)-galactosyltransferases, which create galactose-beta(1, 4)-N-acetylglucosamine linkages. One model is therefore that SQV-8 uses a SQV-3 product as a substrate. SQV-7 is similar to members of a family of nucleotide-sugar transporters. The sqv genes therefore are likely to encode components of a conserved glycosylation pathway that assembles a C. elegans carbohydrate moiety, the absence of which perturbs vulval invagination.

Specific inhibition of an alpha-galactosyltransferase from Trypanosoma brucei by synthetic substrate analogues.

Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.

Enzymatic assay of galactosyltransferase by capillary electrophoresis.

The kinetic parameters of a galactosyltransferase-catalyzed reaction were determined for the first time using capillary zone electrophoresis (CZE) using the methylumbelliferyl (MU) glycoside of N-acetylglucosamine as the acceptor molecule. The CZE was performed using borate buffer and the enzymatic transformations were monitored at 214 nm. The kinetic parameters obtained for MU-GlcNAc were Km = 35.9 microM and Vmax = 7.5 micromol/min/mg, and those for UDP-Gal were Km = 115.3 microM and Vmax = 12.4 micromol/min/mg. A representative inhibition assay was also carried out using UDP as an inhibitor to give the Ki value of 83.9 microM against MU-GlcNAc. The structure of the synthetic product was also confirmed using 1H NMR spectroscopies after isolation by simple chromatography.

Differential effect of alpha-lactalbumin on beta-1,4-galactosyltransferase IV activities.

We isolated a human cDNA clone encoding beta-1,4-galactosyltransferase (beta-1,4-GalT IV) which shares 37% identity with previously characterized mammalian beta-1,4-GalT (beta-1,4-GalT I). By transfection of the full length cDNA into Sf-9 cells and assay of the cell homogenates, higher beta-1,4-GalT activity toward GlcNAc beta-S-pNP was obtained, and its activity was modulated with alpha-lactalbumin, while no lactose synthetase activity was detected in the presence of alpha-lactalbumin. Northern blot analysis using total and poly (A)+ RNA preparations revealed that the expression level of beta-1,4-GalT IV transcript is low and relatively constant while that of beta-1,4-GalT I transcript is dramatically increased in the mouse mammary gland during lactation. These results indicate that beta-1,4-GalT IV can interact with alpha-lactalbumin but has no lactose synthetase activity.

Development of an enzyme-linked immunosorbent assay-based method for measuring galactosyltransferase activity using a synthetic glycopolymer acceptor substrate.

A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring beta 1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a beta-linked N-acetylglucosamine (GlcNAc beta) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, beta-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by using Ricinus communis agglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAc beta), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.

Beta1,4-galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function.

Beta1,4-galactosyltransferase (beta4GalT-I) is a constitutively expressed trans-Golgi enzyme, widely distributed in vertebrates, which synthesizes the beta4-N-acetyllactosamine structure commonly found in glycoconjugates. In mammals beta4GalT-I has been recruited for a second biosynthetic function, the production of lactose; this function takes place exclusively in the lactating mammary gland. In preparation for lactose biosynthesis, beta4GalT-I enzyme levels are increased significantly. We show that mammals have evolved a two-step mechanism to achieve this increase. In step one there is a switch to the use of a second transcriptional start site, regulated by a stronger, mammary gland-restricted promoter. The transcript produced is distinguished from its housekeeping counterpart by the absence of approximately 180 nt of 5'-untranslated sequence. In step two, this truncated transcript is translated more efficiently, relative to the major transcript expressed in all other somatic tissues.

Mammary sensitivity to protein restriction and re-alimentation.

The present study tested the influence of protein undernutrition and re-alimentation on mammary gland size and secretory cell activity in lactating rats. During gestation, female Sprague-Dawley rats were offered a high-protein diet (215 g crude protein (N x 6.25; CP)/kg DM; H); litters were standardized to twelve pups at parturition. During lactation, two diets were offered ad libitum, diet H and a low-protein diet (90 g CP/kg DM; L). Lactational dietary treatments were the supply ad libitum of either diet H (HHH) or diet L (LLL) for the first 12 d of lactation, or diet L transferring to diet H on either day 6 (LHH) or 9 (LLH) of lactation. On days 1, 6, 9 and 12 of lactation, rats from each group (n > or = 6) were used to estimate mammary dry mass, fat, protein, DNA and RNA; the activities of lactose synthetase (EC 2.4.1.22) enzyme and Na+,K(+)-ATPase (EC 3.6.1.37) were also measured. Rats offered a diet considered protein sufficient (H) from day 1 of lactation showed a decrease in mammary dry mass and fat but an increase in DNA, RNA and protein on day 6, after which there was no further change, except for mammary protein which continued to increase. However, rats offered diet L showed a steady loss in mammary mass and fat throughout the 12 d lactation period and no change in mammary DNA, RNA or protein. Rats previously protein restricted for either the first 6 or 9 d of lactation had their mammary dry mass and mammary fat loss halted and showed a rapid increase in mammary DNA, RNA and protein on re-alimentation. Lactose production in group HHH, as measured by lactose synthetase activity, was similar on days 1 and 6 of lactation, after which a significant increase was seen. Protein-restricted rats showed no change in lactose synthetase activity during the 12 d experimental period. Changing from diet L to diet H led to a significant increase in lactose synthetase activity to levels comparable with those offered diet H from day 1. These results show that rats offered a protein-restricted diet during lactation suffer mammary underdevelopment, but this may be rapidly reversed by re-alimentation with a high-protein diet.

Membrane-bound states of alpha-lactalbumin: implications for the protein stability and conformation.

alpha-Lactalbumin (alpha-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC alpha-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-alpha-LA was sensitive to two thermal transitions: the Tc of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 degrees C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-alpha-LA complex showed a distinct fluorescence thermal transition between 40 and 60 degrees C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.

Biosynthesis in vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core.

The galactosyltransferase, GalT-4, which catalyses the biosynthesis in vitro of neolactotetraosylceramide, nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (Glc NAc beta 1-3Gal beta 1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc beta 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn2+). The K(m) values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 microM, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-alpha 1-acid glycoprotein or N-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc beta 1-1-3Gal beta 1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for an N-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH2 beta 1-3Gal beta 1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while the K(m) and Vmax of the reacetylated substrate (GlcNAc beta 1-3Gal beta 1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.