Hexosamine Moiety Research Articles

Identification and characterization of glycoproteins on the spore surface of Clostridium difficile.

In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-β-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-β-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the β-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages.

Semiquantitative analysis of isomeric oligosaccharides by negative‐ion mode UV‐MALDI TOF postsource decay mass spectrometry and their fragmentation mechanism study at N‐acetyl hexosamine moiety

Postsource decay (PSD) spectra of isomeric neutral lactooligosaccharide mixtures were measured from the chlorinated molecules [M + Cl]- by negative-ion mode ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) to estimate quantitatively the mixing ratios in their mixtures. The PSD ions specific to each isomeric structure were used to distinguish the linkage and branching isomers, and the molar ratios of the isomers were estimated from their ion abundances. The relative ion abundances changed linearly in the PSD spectra of the mixtures of the isomers as their molar ratio was varied in the analyte solutions. Therefore, the molar ratios of the isomers in the analyte mixtures could be estimated semiquantitatively. In addition, we studied their fragmentation mechanisms in N-acetyl hexosamines such as GlcNAc, which enabled us to quantitatively analyze the structures of the isomers of lactooligosaccharides. The conjugated systems elongate in the chemical species of the Z-type fragmentation on the 3-linked GlcNAc owing to the acetoamido groups at the C-2 positions, which made the chemical species of the Z-type ions stable. The glycosyl bonds of the front of GlcNAc cleaved easily as a C-type fragmentation because the negative charge at the anomeric position could be delocalized to the carbonyl oxygen atom at the acetoamido group of GlcNAc. These factors caused the stabilization of the chemical species of the C/Z fragment ions produced by the double cleavage around GlcNAc.

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli and 2-O-desulfated heparin.

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.

Glycolipids isolated from cultured rat hepatocytes: Analysis of their role in insulin signal transduction

The precise mechanism by which insulin elicits its effects remains to be fully determined. A glycophospholipid, isolated from H35 cells, has been proposed as a possible precursor for an insulin-generated second messenger that mediates the intracellular effects of insulin. This glycolipid contains a hexosamine moiety, inositol, galactose and palmitate. We have isolated a glycolipid from cultured rat hepatocytes that exhibits chromatographic and radiolabelling characteristics similar to this proposed precursor. The glycolipid can be radiolabelled with glucosamine, galactosamine and palmitate, but not myristate or myo-inotisol. Incorporation of radiolabel into this glycolipid was insensitive to the presence of either insulin (10 −7 M) or phosphatidylinositol-specific phospholipase C (PI-PLC) in the culture medium. The cultured hepatocytes used exhibited normal insulin responses with respect to glycogen turnover and gene expression. Treatment of partially purified glycolipid with either PI-PLC or nitrous acid did not result in the generation of an aqueous soluble phosphooligosaccharide indicating that the glycolipid was not cleaved by either agent. This is in contrast to the reported cleavage of the glycolipids found in H35 hepatoma and lymphocytes. These results question the role of the putative phosphooligosaccharide mediator in the intracellular transduction system activated by insulin.

Isolation and characterization of proteoglycans synthesized by cultured mesangial cells.

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.

Chemical effects of UV radiation on aqueous solutions of heparin-Ca salt

Chemical effects of 184·9 nm, 194·2 nm and 253·7 nm ultraviolet radiation on dilute aqueous solutions of heparin-Ca salt are investigated. The degradation of heparin in dilute aqueous solutions involves two different mechanisms: (a) at 184·9 nm, an H • abstraction process takes place: and (b) at 194·2 nm and 253·7 nm, heparin free radicals formed due to glycosidic bond dissociation are involved. These mechanisms are responsible for the depolymerisation of heparin molecules. The presence of sulphate groups renders stability to heparin, which requires long exposure to ultraviolet radiation to produce metachromasia in the cationic dye Acridine Orange, and changes in reducing-end group, hexuronic acid and hexosamine moieties in the presence of oxygen or nitrogen. The effects of three wavelengths and the existence of two degradation pathways are discussed.

Chemical effects of UV radiation on aqueous solutions of chondroitin-6-sulphate

Ultraviolet radiation degradation of dilute aqueous solutions of chondroitin-6-sulphate has been investigated using 184·9 nm, 194·2 nm and 253·7 nm radiations. Two different pathways are involved. The degradation proceeds via H • atom abstraction with 184·9 nm radiation and via chondroitin-6-sulphate free radicals with 194·2 nm and 253·7 nm radiation, rupturing glycosidic linkages, as indicated by depolymerization of the polysaccharide. Depolymerization produces changes in metachromasia of Acridine Orange, reducing-end group, hexuronic acid and hexosamine moieties of chondroitin-6-sulphate. No release of sulphate was observed. The effects of the three wavelengths on these parameters in an oxygen or nitrogen atmosphere and the existence of two degradation pathways are discussed.

On the structure of heparitin sulfates Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.

Studies on bio-flocculation with the flocculants produced by microorganisms. II. Purification and chemical analysis of microbial cell flocculant produced by Aspergillus sojae AJ7002.

A bio-flocculant was isolated from the culture broth of Asp. sojae AJ 7002. It was partially purified by acetone or ethanol precipitation, by ion-exchange and gel chromatography, and by dialysis. The isolated polymer possessed chemical characteristics of a poly-hexosamine and a protein. Glucosamine and galactosamine were not acetylated. The flocculant contained 2-ketogluconic acid, but sulfur or phosphorus was not detected. This flocculant was thermo-stable and its activity varied with pH. It was suggested that the hexosamine moiety in the polymer played a major role in bio-flocculation, assisted by protein portion in enlargement of the molecular weight of the flocculant, and by 2-ketogluconic acid in endowing it with amphoteric character.

Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues.

L-[14C]Iduronic acid-containing sulfated galactosaminoglycans were formed by incubation of a fibroblast particulate fraction with UDP-D[14C]glucuronic acid, UDP-N-acetylgalactosamine, and sulfate donor (3'-phosphoadenylylsulfate). The formation of L-iduronic acid was strongly promoted by concomitant sulfation of the polymer. In the absence of sulfate donor 5 to 10% of the [14C]uronic acid residues were L-iduronic acid. However, when 3'-phosphoadenylylsulfate was included in the incubation mixture the amount of L-iduronic acid in the product increased 3 to 5-fold. Furthermore, approximately the same quantity of L-[14C]iduronic acid was recovered from the product formed in a pulse-chase experiment where incorporation of 14C-isotope preceded sulfation. It was therefore concluded that C-5 inversion of D-glucuronic acid to L-iduronic acid occurred on the polymer level as shown previously for the biosynthesis of heparin (Hook, M., Lindahl, U., Backstrom, G., Malmstrom, A., AND Fransson, L-A., J. Biol. Chem. (1974) 249, 3908). This conclusion was supported by the finding that no L[14C]iduronic acid could be detected in the UDP-hexuronic acid pool during this experiment. Nonsulfated and sulfated [14C]galactosaminoglycan products were degraded separately with chondroitinase-AC. The non-sulfated products afforded primarily disaccharide and a small amount of tetrasaccharide, while the sulfated products yielded, in addition, a considerable amount of larger oligosaccharides. Tetrasaccharides from nonsulfated products contained L-iduronic acid indicating that C-5 inversion at solitary sites can occur in the absence of sulfation of adjacent hexosamine moieties. The larger oligosaccharides obtained after chondroitinase-AC digestion of sulfated products yielded L-iduronic acid upon acid hydrolysis and were susceptible to chondroitinase-ABC digestion. The split products were almost exclusively 4-sulfated disaccharides. These results demonstrate that formation of blocks of L-iduronic acid-containing repeat periods is associated with 4-sulfation of adjacent hexosamine moieties.

The copolymeric structure of pig skin dermatan suplhate. Characterization of D-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate.

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.

Glycoprotein biosynthesis: VI. Regulation of uridine diphosphate [formula omitted] metabolism in bovine thyroid gland slices

1. 1. Bovine thyroid gland slices were incubated with 6-diazo-5-oxo- L-norleucine (DON) and in many cases along with trace amounts of either [ 14C]glucosamine or [ 14C]fructose. DON caused a marked inhibition (approx. 95% in 4 h) of L-glutamine: D-fructose -6-P amidotransferase, the enzyme believed to be responsible for the de novo synthesis of glucosamine-6- P, and hence of UDP- N-acetylhexosamines. 2. 2. The reduction of the amidotransferase activity by DON was also demonstrated by measuring the incorporation of [ 14C]fructose into the hexosamine moiety of UDP- N-acethylhexosamines. This incorporation was inhibited by about 91% in 4 h incubation. 3. 3. However, in contrast to previous studies (Bates, C. J., Adams, W. R. and Handschumacher, R. E. (1966) J. Biol. Chem. 241, 1705; Bates, C. J. and Handschumacher, R. E. (1969) Adv. Enzyme Regul. 7, 183) with rat liver in vivo, where there was an 85% reduction of the level of UDP- N-acethylhexosamines by DON, this same inhibitor showed no effect on the concentration of UDP- N-acethylhexosamines in the thyroid slices. This result strongly suggested that in the thyroid, the intracellular hydrolysis of thyroglobulin appreciably contributed N-actylglucosamine to the maintenance of UDP- N-acetylhexosamines pool. 4. 4. DON was shown to have no effect on the conversion of [ 14C]glucosamine to UDP- N-acetylhexosamines and the subsequent utilization of the aminosugar nucleotide for thyroglobulin or glycoprotein biosynthesis. 5. 5. The feedback inhibition of the amidotransferase by UDP- N-acetylglucosamine was demonstrated in the thyroid slices by incubation with puromycin, a well-known inhibitor of protein biosynthesis, which prevented the utilization of the aminosugar nucleotide for the formation of glycoproteins.

NMR Analysis on Acetamide and N-Methyl Groups of Mucopolysaccharides

N-Acetate-methyl signals were examined in the NMR spectra of a series of mucopolysaccharides and some glycopeptides in reference with those of the component monosaccharides involving axial or equatorial acetamide group. The signals showed no significant change on the chemical shifts and shaps in 8 m urea, 1 n NaOH, 1 n HCl and D2O. The signals appeared in δ 2.00 to 2.06 ppm which fall in equatorial region. These results indicate that hexosamine moieties in these polymers keep the C 1 conformation in solutions. A deshielding effect of N-methyl group on the N-acetate-methyl signals was evident with N-methylated mucopolysaccharides.

Chemical effects of -irradiation of aqueous solutions of chondroitin-4-sulphate.

SummaryAfter γ-radiolysis of de-aerated aqueous solutions of sodium chondroitin 4-sulphate, acids (G=2·6) and reducing products (G=0·4) are readily produced. The yields of these products are approximately doubled when the irradiation is carried out in N2O-saturated solution. Depolymerization of the polymer is accompanied by a decrease in the ability of the polymer to bind to methylene blue and cetyl pyridinium chloride, and at a dose of 11 × 1018 eV ml−1 in argonsaturated solution, five low molecular weight products, the smallest of which approximates to a tetramer, are readily discernable. Analysis of the products indicates that the hexosamine moiety is more stable towards ionizing radiations than the hexuronic acid.

The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage.

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-(14)C]glucose and [(35)S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride-cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-(14)C]glucose were found to be 0.79+/-0.16 and 3.2+/-0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-(14)C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [(35)S]sulphate incorporation and were found to be in good agreement with those obtained from [U-(14)C]glucose labelling.

NMR study of 4‐deoxy‐α‐L‐threo‐4‐enohexopyranosyluronic acid (1 → 3)2‐acetamido‐2‐deoxy‐D‐hexoses produced in the enzymic digestion of hyaluronate, chondroitin and chondroitin sulfates

AbstractThe conformation of 4‐deoxy‐α‐L‐threo‐4‐enohexopyranosyluronic acid (1 → 3)2‐acetamido‐2‐deoxy‐d‐hexoses was analyzed by NMR spectroscopy. The half‐chair (H12) conformation was found in the unsaturated uronide moiety and the chair (Cl) conformation in the hexosamine moiety.

Studies on the incorporation of (U-14C)glucose and (35S)sulphate into the acid glycosaminoglycans of neonatal rat skin.

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.

Hexosamine intermediates in the human gastric mucosa

The proctection of human gastric mucosa depends on glycosaminoglycans and glycoproteins, which are not digested by pepsin and hydrochloric acid (1). Our data concern the enzyme, present in human gastric mucosa, which synthesises glucosamine 6-phosphate, the first glucosamine intermediate in biosynthesis of macromolecular substances. Recently Kent and Allen (2) have found that human gastric mucosa is able to incorporate the radioactivity from glucose into bound in glycosaminoglycans and glycoprotein biosynthesis in human gastric mucosa have not been described. This article concers the detection and characterization of glucosamine intermediates in human gastric mucosa and elucidates the possible routes of the biosynthesis of hexosamine moiety in the human gastric secretion.

Reactions of connective tissue and related polyanions with hydrated electrons and hydroxyl radicals

The effects of ionizing radiations on aqueous solutions of connective tissue and other polyanions have been examined by using pulse radiolysis. Rate constants have been measured for the reactions of hydrated electrons and OH radicals with chondroitin 4-sulphate, chondroitin 6-sulphase, N-desulphated heparin, sulphated hyaluronic acid, keratan sulphate, and sodium dextran sulphate, and the results are related to previous measurements on heparin and hyaluronic acid. The results indicate that it is the presence of the hexosamine moiety which is responsible for the enhanced reactivity of certain polyanions towards hydrated electrons, compared with unsubstituted carbohydrates. However, although there is significant reaction with hydrated electrons, it is the OH radicals which exert the main contribution towards the chemical changes induced by irradiation in aqueous solution. Consideration of the nature of the spectra of the transient species produced after pulsed irradiation support this conclusion.

Separation of N-acetyl-alpha-glucosaminidase and N-acetyl-alpha-galactosaminidase from ox spleen. Cleavage of the O-glycosidic linkage between carbohydrate and polypeptide in ovine and bovine submaxillary glycoprotein by N-acetyl-alpha-galactosaminidase.

1 Two specific N-acetyl-α-hexosaminidases were detected in bovine spleen homogenates, purified by ammonium sulphate precipitation, separated by gel filtration and identified as N-acetyl-α-glucosaminidase and N-acetyl-α-galactosaminidase respectively. In contrast to the established N-acetyl-β-hexosaminidase which splits phenyl-β-d-glycosides of both N-acetyl-glucosamine and N-acetylgalactosamine, the two N-acetyl-α-hexosaminidases exhibits an absolute specificity towards the hexosamine moiety of their substrates. N-Acetyl-α-galactosaminidase acts on phenyl-N-acetyl-α-d-galactosaminide and not on the corresponding glucosaminide. The reverse holds for N-acetyl-α-glucosaminidase. 2 In the cell-free supernatant of a bovine spleen homogenate N-acetyl-α-galactosaminidase is present with a specific activity about 50-fold of that of the corresponding glucosaminidase, using the appropriate phenyl-α-d-hexosaminides as substrate. 3 Ox spleen N-acetyl-α-galactosaminidase was found to split both phenyl-N-acetyl-α-d-galactosaminide and the O-glycosidic linkage between terminal N-acetylgalactosamine and peptide-bonded serine (threonine) in ovine and bovine submaxillary glycoproteins. The identity of bovine O-seryl-N-acetylgalactosaminide glycosidase and N-acetyl-α-galactosaminidase is clearly demonstrated by the presence of these enzymatic activities in a single chromatographic peak even after re-chromatography. 4 One substrate of natural origin for N-acetyl-α-glucosaminidase was found in UDP-N-acetylglucosamine.