Galactose-6-sulfatase from Actinobacillus sp. IFO-13310 and its action on sulfated oligosaccharides from keratan sulfate.

A 6-sulfatase specific for sugasr of the galactose configuration was purified 81-fold from the crude extract of Actinobacillus sp. IFO-13310. This preparation contained activity towards both N-acetylgalactosamine 6-sulfate and galactose 6-sulfate (relative activity, 2.4 : 1). The enzyme also release inorganic sulfate from the non-reducing galactose 6-sulfate end group of a trisaccharide disulfate prepared from keratan sulfate by sequential degradation with endo-beta-galactosidase, N-acetylglucosamine-6-sulfatase and exo-beta-N-acetylglucosaminidase. In addition, a tetrasaccharide trisulfate bearing the non-reducing N-acetylglucosamine 6-sulfate end group, also enzymatically prepared from keratan sulfate, was degraded to give rise to inorganic sulfate, N-acetylglucosamine and galactose by the sequential action of this enzyme, N-acetylglucosamine-6-sulfatase, exo-beta-N-acetylglucosaminidase and exo-beta-galactosidase (Charonia lampas).     

Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation

Conditions were established for desulphation of hexa-, octa-, deca- and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo-beta-galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic-plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide-lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5-D-4, 1-B-4 and MZ15, but they mask the i antigen activity of the linear poly-(N-acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after beta-N-acetylglucosaminidase treatment of desulphated linear hexa-, octa- and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti-i antibody, Den, there is an absolute requirement for the non-reducing beta-galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti-i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N-acetylglucosamine residue at the non-reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5-D-4, have provided the first evidence for the presence of glucosamine residues that may be N-sulphated in corneal keratan sulphate.     

Degradation of keratan sulphate by beta-N-acetylhexosaminidases A and B.

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay--Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH--activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.     

Characterization of purified human liver acid beta-D-galactosidases A2 and A3.

1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Structural features of the sulphated polysaccharide from a green seaweed,Caulerpa taxifolia

A sulphated heteropolysaccharide was isolated from a green seaweed, Caulerpa taxifolia, by extraction with acid and purified via its copper complex. Methylation analysis of both the sulphated and desulphated polysaccharides revealed the presence of 1,4-linked xylose, 1,6-linked galactose, 1,4,6-linked mannose and non-reducing galactose end group units which are all devoid of sulphate groups. In addition 1,4-linked galactose units sulphated at C-3 are also present. Quantitative periodate oxidation showed a consumption of 1.30 and 1.60 moles of oxidant per anhydrosugar unit in the sulphated and desulphated polysaccharides respectively. The oxo-polysaccharides after reduction and hydrolysis revealed the presence of glycerol, erythritol and unoxidized galactose in the mol ratio 11.6:5.1:4.9 and 11.2:5.0:1.0 respectively, besides threitol (3.9 mol) in the desulphated polysaccharide.     

Human serum contains N-acetyllactosamine: beta 1-3 N-acetylglucosaminyltransferase activity

Human serum was shown to contain N-acetyllactosamine: N-acetylglucosaminyltransferase activity. The reaction product was hydrolyzed by beta-N-acetylglucosaminidase and released [14C]N-acetylglucosamine, indicating that the N-acetylglucosaminyl residue was beta-linked to N-acetyllactosamine. Methylation and hydrolysis of the reaction product yielded 2,4,6-trimethyl[3H]galactose, indicating that the N-acetylglucosaminyl residue was introduced at position C-3 of the terminal galactose of N-acetyllactosamine. In our experiments, 2,3,4-trimethyl[3H]galactose was not detected. Substrate competition studies between N-acetyllactosamine and lactose showed that this enzyme also catalyzed the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to lactose. Since the Km value for N-acetyllactosamine, which was 7.0 mM, was approximately a fourth of that for lactose (29.8 mM), N-acetyllactosamine was more effective than lactose as an acceptor.     

A fused lobes gene encodes the processing beta-N-acetylglucosaminidase in Sf9 cells

Manalpha6(Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual beta-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Manalpha6(GlcNAcbeta2Manalpha3)Manbeta4GlcNAcbeta4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing beta-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing beta-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum beta-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing beta-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing beta-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing beta-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum beta-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation.     

Production of a sialylated N-linked glycoprotein in insect cells: role of glycosidases and effect of harvest time on glycosylation

Using a nonengineered Trichoplusia ni insect cell line, Tn-4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N-acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner-flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post-infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of beta-galactosidase and N-acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular beta-galactosidase and N-acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn-4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.     

The enzymic degradation of ovalbumin and its glycopeptides

1. Ovalbumin glycopeptides, freed from all amino acids other than aspartic acid and a small proportion of leucine by repeated digestion with Pronase, were hydrolysed by 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (glycoaspartamidase) to the corresponding oligosaccharides. The glycoaspartamidase did not attack ovalbumin itself. 2. Ovalbumin, with mannose/hexosamine ratio 5:4, lost 1.5moles of N-acetylglucosamine and more than 2moles of mannose after incubation with alpha-mannosidase and beta-N-acetylglucosaminidase respectively. 3. In ovalbumin glycopeptides with approximate mannose/hexosamine ratios 5:3 and 5:4, one and two N-acetylglucosamine residues respectively were accessible to the action of beta-N-acetylglucosaminidase. 4. A mixture of alpha-mannosidase and beta-N-acetylglucosaminidase, acting on an ovalbumin glycopeptide with mannose/hexosamine ratio 5:3.7, removed nearly 4moles of mannose and 1.5moles of N-acetylglucosamine. 5. alpha-Mannosidase removed about 1.5moles of mannose from the ovalbumin oligosaccharide with mannose/hexosamine ratio approx. 5:3. The subsequent action of beta-N-acetylglucosaminidase liberated less than 1mole of N-acetylglucosamine and made at least 1mole further of mannose accessible to alpha-mannosidase action. 6. It is concluded that the carbohydrate moiety of ovalbumin is linked through a glycosyl group to asparagine. In a molecule with mannose/hexosamine ratio 5:4, there are two beta-N-acetylglucosamine residues linked together in a terminal position, followed by alpha-mannose. There is also present a side chain containing two alpha-mannose units.     

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.     

Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells

Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.     

Inhibition of Charonia lampas ascorbate-2-sulfate sulfohydrolase activity by adenosine 5'-diphosphate and related compounds.

1) ADP was a potent inhibitor of the ascorbic-2-sulfate sulfohydrolase activity of Charonia lampas liver. The inhibition was competitive with respect to ascorbate 2-sulfate. The Ki value was 5.9 muM. ADP did not inhibit arylsulfatase (EC 3.1.6.1) of the same organism. 2) Other nucleoside 5'-diphosphates and GTP showed similar inhibition of ascorbate-2-sulfate sulfohydrolase activity. 3) The effects of different nucleosides, nucleotides, and sugar phosphates on ascorbate-2-sulfate sulfohydrolase activity were investigated. Phosphate derivatives other than 3',5'-cyclic AMP were more or less inhibitory.     

Glycosidases in normal and regenerating chicken liver, hepatoma Mc-29, Rous sarcoma, in turkey poult liver and hemocytoblastomes, provoked by the leukosis virus strain Mc-31

Four glycosidases (beta-galactosidase, alpha-mannosidase, alpha-fucosidase and beta-N-acetylglucosaminidase) were studied in chicken normal and regenerating liver, in turkey poult liver and in virus induced avian tumors--chicken hepatoma (strain Mc-29), Rous sarcoma (strain Schmidt-Ruppin) and turkey poult hemocytoblastoma nodules (strain Mc-31). The multiple forms of beta-N-acetylglucosaminidase were assayed as well. A particular enzyme pattern was found in the tumor lines under investigation. A characteristic property of hepatoma cells was the elevation of beta-galactosidase activity and of the former enzyme and that of beta-N-acetylglucosaminidase for the hemocytoblastoma. In Rous sarcoma the glycosidase activities (except that of alpha-fucosidase) were much lower, compared to the other two solid tumors. All enzyme activities were compared with those in the normal liver of the corresponding avian species, and with the liver of tumor bearing fowls and with regenerating chicken liver. Unlike the rat liver in the avian normal and tumor tissues the percentual ratio between the multiple forms A and B of beta-N-acetylglucosaminidase was found to be 30:70%.     

Sulphate turnover of surface proteoglycans in cultured rat smooth muscle cells

Turnover of radioactive sulphate-labelled proteoglycans in cultured rat smooth muscle cells was detected by pulse chase techniques. The degradation appeared to take the form of desulphation of sulphated macromolecules, with a loss in total sulphate of approximately 50% in 5 days. The desulphation process occurred in the pericellular/matrix compartment of the culture system and was unaffected by inhibition of matrix formation by beta-aminopropionitrile, or by incubation of cells with lysomotropic inhibitors. There was no evidence for further degradation of desulphated species even when exogenous, radio-labelled proteoglycans were added to fresh cultures and incubated for four days. Labelled macromolecules initiated on xyloside acceptors were desulphated by rat smooth muscle cell cultures more slowly than intact proteoglycans.     

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.     

Impaired degradation of keratan sulphate by Morquio A fibroblasts.

Upon incubation of keratan [35S]sulphate with normal fibroblasts both [35S]sulphate and N-acetylglucosamine 6-[35S]sulphate are liberated. From the products obtained after digestion with various mutant fibroblasts and with purified N-acetylgalactosamine 6-sulphate sulphatase we suggest that (i) [35S]sulphate is released almost exclusively from galactose 6-sulphate residues; (ii) N-acetylgalactosamine 6-sulphate sulphatase exhibits galactose 6-sulphate sulphatase activity; (iii) both sulphatase activities are deficient in Morquio disease type A.     

Effect of Lytic Enzymes of Acanthamoeba castellanii on Bacterial Cell Walls

Extracts of Acanthamoeba castellanii (Neff) contain alpha- and beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, amylase, and peptidase. All of these activities are optimal between pH 3 and 4. These extracts also were found to clarify suspensions of cell walls from nine different gram-positive bacteria, including Micrococcus lysodeikticus. The pH optimum for the lytic activity was between 3 and 4. The extent of lysis of the various cell walls did not correlate with the release of free amino groups and of free N-acetylated sugars from the walls during digestion with these extracts. Suspensions of cell walls of Escherichia coli (a gram-negative bacterium), Cordiceps militaris (a fungus), and Acanthamoeba cysts, as well as of colloidal chitin, were not clarified by incubation with these extracts, although reducing sugars were released from each of these materials. Exhaustive digestion of M. lysodeikticus walls by lysozyme released no free N-acetylglucosamine. The products of exhaustive digestion of this cell wall with Acanthamoeba extracts were free N-acetylglucosamine, free N-acetylmuramic acid, glycine, alanine, glutamic acid, lysine, and N-acetylmuramic acid peptide fragments. These results suggest that the amoeba extracts contain endo- and exo-hexosaminidases, in addition to beta-hexosaminidase and peptide hydrolases.     

Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9.

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.     

Biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine

The biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine has been demonstrated using membrane preparations from pig trachea. Unlike the UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase, which is inhibited by high levels of N-acetylglucosamine, the UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase shows no inhibition at 200 mM N-acetylglucosamine. About 80% of the total disaccharide synthesized at 200 mM N-acetylglucosamine was base-labile suggesting the 1,3-linkage, alpha-Lactalbumin inhibits galactose incorporation into galactosyl-beta 1,4-N-acetylglucosamine but has little or no effect on the activity of the 1,3-galactosyltransferase. Escherichia coli beta-galactosidase readily hydrolyzed the base-stable product, but not the base-labile component. The apparent 1,3-linked disaccharide was reduced with NaBH4 and was isolated by Bio-Gel P-2 column chromatography. Methylation analysis by gas chromatography/mass spectrometry showed tetramethyl galactose and a 3-substituted N-acetylglucosaminitol. Neither the beta 1,4 nor the beta 1,3 disaccharide was hydrolyzed by green coffee bean alpha-galactosidase. Both disaccharides were readily hydrolyzed by bovine testes beta-galactosidase. This is the first report on the galactosyltransferase which catalyzes the synthesis of the galactosyl-beta 1,3-N-acetylglucosamine linkage such as found in the Type I chain of human blood group substances. A tissue survey in rats showed only rat intestine to have readily detectable UDP-galactose: N-acetylglucosamine 3 beta-galactosyltransferase activity. The intestinal membrane fraction like the tracheal enzyme catalyzes the synthesis of two disaccharides as judged by base treatment, and these appear to be the beta 1,3 and beta 1,4 isomers of galactosyl-N-acetylglucosamine.