Accelerated enzymatic galactosylation of N-acetylglucosaminolipids in lipid microdomains.

A fluoro-tagged N-acetylglucosamine-capped glycolipid that can form lipid microdomains in fluid phospholipid bilayers has been shown to be enzymatically galactosylated by bovine ß(1,4)-galactosyltransferase. MALDI MS, HPLC, and LC-MS revealed that the rate of enzymatic transformation was significantly enhanced by lipid clustering; at a 1% mol/mol loading, clustered glycolipids were galactosylated 9-fold faster than glycolipids dispersed across the bilayer surface. The transformation of the GlcNAc "glycocalyx" into a Gal(ß1-4)GlcNAc "glycocalyx" relabeled these vesicles, making them susceptible to agglutination by Erythrina cristagalli lectin (ECL). The kinetic parameters for this transformation revealed a lower apparent Km when the substrate lipids were clustered, which is attributed to multivalent binding to an extended substrate cleft around the active site. These observations may have important implications where soluble enzymes act on substrates embedded within cellular lipid rafts.

Probing elongating and branching ß-D-galactosyltransferase activities in Leishmania parasites by making use of synthetic phosphoglycans.

Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [-6)Gal(ß1-4)Man(α1-OPO(3)H-]. The repeat structures are assembled by sequential addition of Manα1-OPO(3)H and ß-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-(3)H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [(3)H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with ß-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating ß-d-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating ß-d-galactosyltransferase and a branching ß-d-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior.

4Pi-microscopy of the Golgi apparatus in live mammalian cells.

We report the applicability of 4Pi-microscopy to live mammalian cells. Controlled interference of the counterpropagating wavefronts is possible despite the slight variations in cellular refractive index. Superresolved 3D-fluorescence imaging is exemplified with the first representation of the Golgi apparatus in a live cell at approximately 100 nm resolution.

Constitutively active PKB/Akt inhibited apoptosis and down-regulated beta1,4-galactosyltransferase 1 in hepatocarcinoma cells.

Beta1,4-galactosyltransferase1 (beta1,4GT1) is localized both in the Golgi complex and on the cell surface. In our previous study, we first reported that beta1,4GT1 was associated with cycloheximide-induced apoptosis in human hepatocarcinoma cells. In this study, we transfected constitutively active protein kinase B (Gag-PKB), a central mediator of anti-apoptotic signals transduced by the PI3-kinase, into SMMC-7721 human hepatocarcinoma cells, and examined its effect on apoptosis and beta1,4GT1 activity. Flow cytometry analysis showed that apoptosis was inhibited in Gag-PKB transfected SMMC-7721 cells. At the same time, beta1,4GT1 mRNA level and enzyme activities were downregulated in these cells, consistent with which, the content of beta1,4 Gal branch in the glycoconjugates was decreased in stably transfected cells. Cotransfection of beta1,4GT1 promoter/luciferase reporter and Gag-PKB decreased the luciferase reporter activity in a dose-dependent manner, indicating that the differences in mRNA levels might be regulated through promoter function. All these findings suggested that changes of beta1,4GT1 activity might be involved in apoptotic pathway in hepatocarcinoma cells.

Deficiency of UDP-galactose:N-acetylglucosamine beta-1,4-galactosyltransferase I causes the congenital disorder of glycosylation type IId.

Deficiency of the Golgi enzyme UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase I (beta4GalT I) (E.C.2.4.1.38) causes a new congenital disorder of glycosylation (CDG), designated type IId (CDG-IId), a severe neurologic disease characterized by a hydrocephalus, myopathy, and blood-clotting defects. Analysis of oligosaccharides from serum transferrin by HPLC, mass spectrometry, and lectin binding revealed the loss of sialic acid and galactose residues. In skin fibroblasts and leukocytes, galactosyltransferase activity was reduced to 5% that of controls. In fibroblasts, a truncated polypeptide was detected that was about 12 kDa smaller in size than wild-type beta4GalT I and that failed to localize to the Golgi apparatus. Sequencing of the beta4GalT I cDNA and gene revealed an insertion of a single nucleotide (1031-1032insC) leading to premature translation stop and loss of the C-terminal 50 amino acids of the enzyme. The patient was homozygous and his parents heterozygous for this mutation. Expression of a corresponding mutant cDNA in COS-7 cells led to the synthesis of a truncated, inactive polypeptide, which localized to the endoplasmic reticulum.

Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi.

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the beta-galactosyltransferase (beta-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-alpha-N-acetyl-glucosaminyltransferase (O-alpha-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the beta-Gal transferase, the O-alpha-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Effects of Zn(II) on galactosyltransferase activity.

The enzyme beta-4-galactosyltransferase (GT) catalyzes the transfer of a galactosyl group from UDP-galactose to N-acetylglucosamine (GlcNAc) on glycoproteins. In the presence of alpha-lactalbumin (alpha-LA), galactosyltransferase catalyzes the transfer of galactose to glucose to yield lactose. It is known that, in the absence of alpha-lactalbumin, Zn(II) competes with Mn(II) for the same binding site(s) in galactosyltransferase, resulting in an increase in the apparent Michaelis constant, Km(app), for Mn(II)-activation of N-acetyllactosamine synthesis. In the presence of alpha-lactalbumin (i.e., lactose synthase), the Mn(II)-activation is biphasic and the initial phase is inhibited by increasing concentrations of Zn(II). The Zn(II) inhibition of lactose synthase plateaus at [Zn(II)]:[alpha-lactalbumin] approximately 1:1, while for N-acetyllactosamine synthesis there is no plateau at all. The results suggest that Zn(II) binding to alpha-lactalbumin effects lactose synthase. Kinetically, Zn(II) induces a decrease in both the Km(app) and Vm for Mn(II), which results in an apparent increase, followed by a decrease, in lactose synthase activity at Mn(II) concentrations below saturation of the first [Mn(II)] binding site. Increasing Zn(II) also decreases Km(app) and Vm for both glucose and UDP-galactose in the lactose synthase reaction with either both Ca(II)- or apo-alpha-lactalbumin, further suggesting novel interactions between Zn(II)-alpha-lactalbumin and the lactose synthase complex, presumably mediated via a Zn(II)-induced conformational change upon binding to alpha-lactalbumin. On the other hand, in N-acetyllactosamine synthesis, Zn(II) only slightly effects Km(app) for N-acetylglucosamine and has essentially no effect on Km(app) or Vm for UDP-galactose.

Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei.

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.

Purification of uridine diphosphate-galactose:glucosyl ceramide, beta 1-4 galactosyltransferase from human kidney.

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from normal human kidney "buffy coat" preparation employing detergent extraction, ultrafiltration, and Sepharose Q column chromatography. On reducing and nonreducing gels, the enzyme resolved into two bands with apparent molecular weights on the order of 60,000 and 58,000, respectively. The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric focusing revealed that the pI of this enzyme is approximately 4.55. Product characterization and substrate specificity studies employing chromatography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major product synthesized by this enzyme was Cer1-1 beta Glc4-1Gal, and Cer1-1 beta Glc was the preferred substrate. Digestion of the 60- and 58-kDa proteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding suggests that the two proteins may be related to each other. Western immunoblot assays revealed that the antibody against UDP-galactose:GlcCer, beta 1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct enzymes. The availability of the enzyme GalT-2 and corresponding antibody will allow functional studies in the near future.

Properties of IgA-binding receptors on murine T cells: relative importance of Fc alpha R, beta-galactosyltransferase and anti-secretory component reactive proteins (ASCP).

Murine T cells and T-cell lines express receptors for the Fc of IgA (Fc alpha R); however, their molecular properties remain to be elucidated. In the present study, we examined three candidate molecules for IgA-binding receptors including Fc alpha R, beta-galactosyltransferase (beta-GT) and anti-secretory component (SC) reactive proteins (ASCP) expressed on T cells which might participate in the binding of different molecular forms of IgA. T-cell lines derived from CD4+ T cells of mouse Peyer's patches (PP) (designated PPT 4-6 and PPT 4-16) and from cloned PP T helper (Th) cell lines (ThHA1 #9 and #10) bound both monomeric and dimeric IgA (mIgA and dIgA), while the fusion partners (BW 5147 and R1.1) did not. In contrast, both Fc alpha R+ and Fc alpha R- cell lines bound to high molecular weight polymeric or aggregated IgA (pIgA). All cell lines reacted with a monoclonal anti-beta-GT (MoAb) and beta-GT enzyme activity was associated with the cell lysates and membrane fractions of all cells tested. The anti-beta-GT MoAb stained a 47-kDa band on immunoblots which was identical to that seen with native enzyme. mRNA analysis with beta-GT cDNA showed that all cell lines constitutively produced enzyme-specific mRNA. Both Fc alpha R+ T cells and Fc alpha R- control cell lines showed cell surface specific beta-GT activity. This is the first study which shows that mouse T cells produce beta-GT. However, Fc alpha R and beta-GT appear to be separate receptors, because Fc alpha R+ T cells bound mIgA and dIgA, and this treatment did not affect staining with biotinylated anti-beta-GT MoAb. Further, preincubation of the Fc alpha R+ cells with anti-beta-GT MoAb did not block mIgA binding. However, the anti-beta-GT MoAb partially blocked binding of pIgA to both Fc alpha R+ and Fc alpha R- T cells, suggesting that beta-GT may be a receptor for pIgA. Others have shown that T cells may bind IgA through a receptor serologically related to SC. We found that antibodies both to human SC and to rat SC specifically bound to both Fc alpha R+ and Fc alpha R- T cells. Further, a 72-kDa band was detected when cell membrane fractions were analysed with these antisera (ASCP) by solid phase immunoisolation technique and immunoblot analysis. The ASCP is not an IgA-binding receptor, since anti-SC did not block either mIgA or pIgA binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Increase in beta-1,4-galactosyltransferase activity during PC12 cell differentiation induced by forskolin and 2-chloroadenosine.

Galactosyltransferase (GALTase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chloroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine PC carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.

Characterization of a murine beta 1-4 galactosyltransferase expressed in COS-1 cells.

We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha-lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage.

Nerve growth factor-induced increase in [3H]thymidine incorporation into parotid and submandibular glands of young rats and its partial blockade by propranolol or partial sialoadenectomy.

Administration of nerve growth factor (NGF) twice daily for 2 days to young rats (11 days old at the time of the initial injection) resulted in an 8.1-fold increase in [3H]thymidine levels of the parotid gland, and a 9.7-fold increase in levels of the submandibular gland when compared to untreated controls. Isoproterenol (ISO), a beta-adrenergic receptor agonist, caused an 8.7-fold increase in [3H]thymidine incorporation into DNA of the parotid gland, and a 10.7-fold increase in [3H]thymidine in the submandibular gland when compared to controls. The increase in thymidine was accompanied by parotid gland enlargement as well as an increase in cell surface beta 1-4 galactosyltransferase, an enzyme whose expression has been associated previously with acinar cell proliferation. Administration of NGF and ISO together were not additive in their effects on the parotid and submandibular glands. The introduction of propranolol, a beta-adrenergic receptor antagonist, completely negated the ISO effects on the salivary glands but was only partially effective in blocking the NGF effects on the glands. An assay of parotid levels of norepinephrine showed NGF treatment to cause an increase in gland-associated levels of neurotransmitter. Removal of the submandibular/sublingual glands prior to administration of ISO prevented the above changes in the parotid gland. NGF administered to partially sialoadenectomized rats was also less effective in inducing parotid gland hypertrophy and hyperplasia. Simultaneous administration of NGF and ISO to the partially sialoadenectomized rats had an additive influence on [3H]thymidine incorporation, galactosyltransferase expression and gland hypertrophy. The results suggest that NGF influences salivary gland cell growth in part through activation of cell-surface beta-adrenergic receptors.

Effect of homeopathic dilutions on subcellular enzymatic activity.

The activity of various inhibitors on several subcellular enzymes was studied. First we determined the inhibitory concentration required to reduce maximum enzymatic activity by 50%, then the effect of various hahnemannian dilutions of the same inhibitory agent was tested. Seven inhibitory agents were tested in this way on seven different enzymatic systems. No effects of these hahnemannian dilutions were shown.

The interaction of bovine milk galactosyltransferase with lipid and alpha-lactalbumin.

The activation of galactosyltransferase (UDPgalactose: N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) by alpha-lactalbumin has been studied at low concentrations of alpha-lactalbumin where the relationship is sigmoidal. The sigmoidal shape of the activation curve was eliminated by neutral lipids such as phosphatidylcholine and phosphatidylethanolamine, detergents such as Triton X-100 or by an aggregated form of alpha-lactalbumin generated by crosslinking alpha-lactalbumin with dithiobissuccinimidylpropionate. It is proposed that these different reagents present a hydrophobic surface to the enzyme which is necessary for lactose synthase activity. In competition experiments, large amounts of alpha-lactalbumin were able to displace lipid from the enzyme as suggested by the loss of the lipid-activating effect in the presence of an excess of alpha-lactalbumin. Optimal lactose synthase activity was obtained when the ratio of lipid/alpha-lactalbumin/enzyme was 60:6:1. The mechanism by which the lipid effect was obtained probably involved a phase transition in the enzyme which was detected as a sharp break in the Arrhenius curve. The presence of phosphatidylcholine abolished the break demonstrating that full activity of the enzyme required both alpha-lactalbumin and lipid.

Effect of hyperthermia on activity of three glycosyltransferases in Chinese hamster ovary cells.

We measured activities of three glycosyltransferases at various times during heat-induced thermotolerance development. Glycosyltransferases are normally located in the Golgi apparatus and catalyze cellular glycosylation reactions. UDP-Gal:N-acetylglucosamine beta 1,4-galactosyltransferase (beta 1,4-GalT) is known to participate in the formation of N-linked glycoproteins; when compared to cell survival, beta 1,4-GalT activity was significantly more heat resistant (50% loss of activity: 80 min, 45 degrees C) and showed little elevation at a time when thermotolerance was fully expressed. However, beta 1,4-GalT activity increased twofold by 24-h postheating when thermotolerance had begun to decay. Activity of beta 1,4-GalT was compared with glycosyltransferase activities that are considered to be specific for O-linked glycoproteins: UDP-Gal:N-acetylgalactosamine-beta 1,3-galactosyltransferase (beta 1,3-GalT), and UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (Gal-NAcT). Heat-inactivation experiments with heating times up to 60 min at 45 degrees C failed to reduce either activity below that of unheated control cells. Instead both beta 1,3-GalT and GalNAcT activity increased approximately twofold immediately after 10 min at 45 degrees C. Activity of beta 1,3-GalT rapidly decreased with time after heating and returned to control levels by 6-h postheating. In contrast, GalNAcT activity continued to increase with time after 10 min at 45 degrees C, and was 4.5-fold above unheated controls by 6-h postheating. GalNAcT activity returned to control levels 24- to 48-h postheating. A comparison with the cellular survival response showed that GalNAcT activity preceded thermotolerance expression by 2-4 h and also decayed more rapidly than heat resistance in thermotolerant cells. These data, together with other published results, suggest that expression of thermotolerance may be associated with enhanced glycosylation of intracellular proteins.

Isoprenaline-induced transcription of 4 beta-galactosyltransferase is inhibited by both cycloheximide and actinomycin D in rat parotid acinar cells.

A cDNA clone for the Golgi enzyme 4 beta-galactosyltransferase (EC 2.4.1.38) was used to determine the steady-state mRNA content in cultured rat parotid acinar cells. Isoprenaline, a beta-adrenergic-receptor agonist, caused an increase in steady-state amounts of mRNA for 4 beta-galactosyltransferase in cultured acinar cells as well as in specific activity of the enzyme. The amount of 4 beta-galactosyltransferase-specific mRNA was dependent on transcription of the gene, as determined by incubation of cells with the RNA polymerase inhibitor actinomycin D, concomitant with the time of isoprenaline treatment. Transcription of the 4 beta-galactosyltransferase gene also required the active biosynthesis of additional cellular factors, since isoprenaline-induced increases in mRNA amounts were not observed on co-incubation with the protein-synthesis inhibitor cycloheximide.

Modulation of two distinct galactosyltransferase activities in populations of mouse peritoneal macrophages.

We have examined two galactosyltransferase activities in membrane preparations obtained from resident macrophages, from resident macrophages maintained in culture for 24 hr, and from thioglycollate (TG)-elicited macrophages. Transfer of galactose from uridine diphosphate (UDP)-galactose to N-acetylglucosamine is 2.6 times higher in membranes prepared from TG macrophages (107 +/- 5.5 nmol/hr/mg) than in membranes prepared from resident macrophages (41 +/- 2.0 nmol/hr/mg). Membranes obtained from resident macrophages cultured for 24 hr exhibit a 2.5 times higher activity (102 +/- 4.4 nmol/hr/mg) than membranes from resident cells plated for 4 hr. Transferase activity in membranes derived from TG macrophages is not significantly affected by overnight culture. The transferase reaction product, isolated on Bio-Gel P-4 and analyzed by galactosidase treatments, was identified as galactosyl-beta 1, 4-N-acetylglucosamine. The enzyme, therefore, is UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase. This is supported by the fact that this galactosyltransferase activity is specifically inhibited by high concentrations of N-acetylglucosamine (200 mM). We have also examined the transfer of galactose to N-acetyllactosamine. Membranes from TG-elicited macrophages contain a UDP-galactose:galactosyl-beta 1, 4-N-acetylglucosamine 3 alpha-galactosyltransferase which synthesizes the trisaccharide, galactosyl-alpha 1, 3-galactosyl-beta 1,4-N-acetylglucosamine. This product was identified by gel filtration chromatography, high performance liquid chromatography, and galactosidase digestions. This alpha-galactosyltransferase activity was not detected in membranes prepared from resident macrophages. These results indicate that glycosyltransferase activities are modulated in populations of mouse macrophages, and that these changes correlate with changes in cell surface lactosaminoglycans reported previously.