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PubMed1976

Sanfilippo disease type B: presence of material cross reacting with antibodies against alpha-N-acetylglucosaminidase.

1. alpha-N-Acetylglucosaminidase, the enzyme deficient in Sanfilippo disease type B (mucopolysaccharidosis III B) was purified from normal human urine. An antiserum was raised in rabbits against the purified enzyme. Preincubation of the antiserum with crude alpha-N-acetylglucosaminidase from normal human urine, followed by centrifugation, led to a marked reduction of the enzyme activity in the supernatant. Formation of the antibody-enzyme complex had no influence on the activity. The thermal stability of the enzyme was markedly enhanced by complex formation with the antiserum. 2. In the urine from three patients with Sanfilippo disease type B the presence of cross-reacting material could be demonstrated by incubating the antiserum with alpha-N-acetylglucosaminidase in the presence of Sanfilippo B urine or by pretreatment of the antiserum with Sanfilippo B urine. 3. Immunodiffusion and immunoelectrophoresis of crude normal or Sanfilippo B urine gave rise to up to four precipitation lines, only one of which exhibited alpha-N-acetylglucosaminidase activity in the case of normal urine. Purified alpha-N-acetylglucosaminidase yielded only a single precipitation line. After adsorption with the purified enzyme the antiserum did not cross react with any of the urinary proteins. 4. On a quantitative determination of cross-reacting material using Sepharose immobilized antibodies in the urine from two Sanfilippo B patients the amount of cross-reacting material appeared to be less than one fourth of the amount of alpha-N-acetylglucosaminidase protein in an age-matched control urine. The cross-reacting material present in the urine of Sanfilippo B patients had a significant lower binding affinity for antibodies against alpha-N-acetylglucosaminidase than preparations from normal human urine. Taking into account this lower binding affinity, it can be calculated that the amount of cross-reacting material in the urine of Salfilippo B patients exceeds that of normal controls. 5. It is concluded that Sanfilippo disease type B is due to a mutation of a structural gene coding for alpha-N-acetylglucosaminidase. The mutation affects the catalytical and immunological properties of the enzyme protein.

Acetylglucosaminidase↗

PubMed1979

Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.

The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.

Acetylglucosaminidase↗

PubMed1978

Human placenta alpha-N-acetylglucosaminidase: purification, characterization and demonstration of multiple recognition forms.

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) was purified from human placenta by a four-step procedure including ammonium sulfate precipitation, affinity chromatography with immobilized antibodies against urinary alpha-N-acetylglucosaminidase, gel chromatography and discontinuous gel electrophoresis with a 30% recovery and 26 300-fold purification. Immunological methods revealed the contamination with about 10% non-alpha-N-acetylglucosaminidase protein. Isoelectric focusing led to a distribution of activity between 4.3 and 6.5 with maxima at pH 5.1 and pH 5.7. After treatment with neuraminidase, alpha-N-acetylglucosaminidase activity assembled at pH 5.7. The multiple isoelectric forms were endocytosed with different rates by cultured human skin fibroblasts. Placenta alpha-N-acetylglucosaminidase has an apparent molecular weight of 304 000 and contains 23.4% carbohydrate consisting of glucose, galactose, mannose, hexosamines and neuraminic acid. Gel electrophoresis in the presence of 0.1% sodium dodecylsulfate separated placenta alpha-N-acetylglucosaminidase into subunits with molecular weights of 86 500 and 81 000. The activity towards various substrates, the kinetics of hydrolysis, the pH optimum and the stability of the catalytic activity were assayed.

Acetylglucosaminidase↗

PubMed1979

Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose.

Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes.

Acetylglucosaminidase↗

PubMed1979

Isolation and characterization of phosphorylated oligosaccharides from alpha-N-acetylglucosaminidase that are recognized by cell-surface receptors.

Adsorptive endocytosis of lysosomal enzymes by fibroblasts and hepatocytes involves binding to cell surface receptors that recognize on lysosomal enzymes a phosphorylated carbohydrate, most likely a mannose 6-phosphate residue [Kaplan et al. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 2026-2030; Ullrich et al. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 1591-1598]. Loss of alpha-N-acetylglucosaminidase endocytosis after treatment with endoglucosaminidase H indicated that the recognition site of alpha-N-acetylglucosaminidase is located on N-glycosidically linked oligosaccharides of the high mannose type. Acidic oligosaccharides with an average molecular weight of 2200 were liberated from alpha-N-acetylglucosaminidase by endoglucosaminidase H. These oligosaccharides were susceptible to degradation by alkaline phosphatase, alpha-mannosidase and beta-N-acetylglucosaminidase. At the non-reducing terminal these oligosaccharides bear phosphorylated mannose and/or N-acetylglucosamine residues.

Acetylglucosaminidase↗

PubMed1976

Retina, tear and serum beta-N-acetylglucosaminidase activities in diabetic patients.

In previous studies, beta-N-acetyglucosaminidase activities were found to be markedly decreased in streptozotocin diabetic rat kidney, while that of the liver, spleen and intestine remained unchanged. The decrease in total kidney enzyme activity was in parallel with a decrease in the enzyme activity of the main isozyme of beta-N-acetylglucosaminidase, of which little or none was contained in the other three tissues. The present paper reports that the retina, also sensitive to diabetic microangiopathy, showed a similar isozyme pattern to that of kidney, composed of mainly type II isoenzyme of beta-N-acetylglucosaminidase. Type II isoenzyme was not detectable in any of the other materials tested including human and rat erythrocytes, lymphocytes and platelets, and human buccal epithelia and saliva, except human tear. The physiologic significance of the human tear beta-N-acetylglucosaminidase is unknown, but this enzyme was found to contain a considerable amount of Type II isoenzyme, and the enzyme activities were decreased in poorly controlled diabetic patients. Patients with retinopathy also showed markedly lowered tear enzyme activity. The diabetic patients were followed up for several months with occasional ophthalmoscopic examination and determination of serum beta-N-acetylglucosaminidase activity. As a result, changes in the latter were found to be useful as an indicator of the development of microangiopathy.

Acetylglucosaminidase↗

PubMed1977

Human alpha-n-acetylglucosaminidase. 2. Activity towards natural substrates and multiple recognition forms.

Purified urinary alpha-N-acetylglucosaminidase acts as an exoglycosidase. The enzyme removes from heparan sulfate exclusively alpha-glycosidically linked N-acetylglucosamine residues. The pH optimum of around 4.4 towards heparan sulfate and heparin is similar to that towards synthetic arylglycosides. Urinary alpha-N-acetylglucosaminidase can be separated by isoelectric focusing into multiple forms with pI values between 3.3 and 6.0. The multiple forms differ in their recognition and endocytosis by cultivated skin fibroblasts. Forms with pI values of 4.8 +/- 0.3 respond best to endcytosis. From these forms up to 0.8 X 10(6) molecules may be recognized and taken up in an hour by a single cell. Sodium periodate treatment reduces the alpha-N-acetylglucosaminidase recognition by fibroblasts and suggests that the recognition sites on the enzyme are associated with its carbohydrate moiety. Attempts to modify the recognition of alpha-N-acetylglucosaminidase by pretreatment with purified glycosidases failed.

Acetylglucosaminidase↗

PubMed1975

Concerted action of beta-glucuronidase and beta-acetylglucosaminidase on hyaluronodextrins.

A kinetic analysis of the stepwise alternating action of beta-glucuronidase and beta-acetylglucosaminidase on oligosaccharides and dextrins derived from hyaluronic acid was undertaken, for better definition of the contribution of this process to hyaluronate catabolism. Production of monosaccharide from larger dextrins by action of either enzyme is powerfully inhibited by electrolyts. In the study, as in mammalian tissues, beta-glucuronidase is present in excess so that the concentration of beta-acetylglucosaminidase is rate controlling in the action on dextrin substrates. For this action, Vmax shows limited variation with ionic strength or molecular weight of substrate. At ionic strength 0.03, but not 0.18, Km decreases some 100-fold for increase of molecular weight from 2,000 to 15,000. It is specifically this decrease in Km that accounts for the prominent electrolyte inhibition observed with larger dextrins. The extremely low values of Km are attributed to multiple ionic enzyme-substrate interactions at sites remote from the catalytic center. The previously reported stimulation by electrolyte of the action of beta-glucuronidase and beta-acetylglucosaminidase on aryl glycosides, studied briefly, is apparently unrelated to the electrolyte effects seen with dextrins. The catabolic contribution of beta-glucuronidase and beta-acetylglucosaminidase appears to be restricted to hydrolysis of the smaller oligosaccharides produced by action of hyaluronidase, since, for any reasonable assumptions regarding cellular environment, the extent of their action on polymeric hyaluronate or larger dextrins must be limited.

Acetylglucosaminidase↗

PubMed1978

Separation of beta-N-acetylglucosaminidase isoenzymes from liver and plasma of six mammalian species by DEAE-cellulose chromatography.

1. The beta-N-acetylglucosaminidase (EC 3.2.1.30) activities of human, pig, calf, lamb, rat and rabbit liver and plasma have been investigated. 2. All preparations had maximum activity between pH 4.0 and 4.5 and Km values with the substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside ranged from 0.54 to 2.54 mM. 3. The isoenzyme profiles of liver and plasma beta-N-acetylglucosaminidase activity were compared using DEAE-cellulose chromatography. In all species the major anionic component of liver (beta-N-acetylglucsaminidase A) was eluted at a higher salt concentration than the most anionic plasma isoenzyme. 4. The plasma beta-N-acetylglucosaminidase A isoenzyme of all species contained sialic acid residues whereas only the rabbit, pig and calf liver isoenzymes were sialylated.

Acetylglucosaminidase↗

PubMed1979

Endocytosis of beta-N-acetylglucosaminidase from sections of mucolipidosis-II and-III fibroblasts by non-parenchymal rat liver cells.

beta-N-Acetylglucosaminidase isolated from the secretions of fibroblasts of mucolipidosis-II and -III patients is internalized by cultured non-parenchymal rat liver cells. The rate of endocytosis compared with that of beta-N-acetylglucosaminidase from control fibroblasts was 11 and 19% for the enzyme from mucolipidosis-II and -III patients respectively. The inhibition of endocytosis by mannan indicates that the beta-N-acetylglucosaminidase from mucolipidosis-II and -III patients is recognized by cell-surface receptors specific for mannose.

Acetylglucosaminidase↗

PubMed1979

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.

Acetylglucosaminidase↗

PubMed1978

[Azoindoxyl methods for the investigation of hydrolases. III. Histochemical studies of beta-D-N-acetylglucosaminidase (author's transl)].

The suitability of various azoindoxyl procedures for the light microscopical demonstration of beta-N-acetylglucosaminidase is described. The incubation media tried consist of 0.5 mg N-Acetyl-(5-bromindol-3-yl)-beta-D-glucosaminide (5-Br-3-indolyl-beta-D-N-acetylglucosaminide; 1 mg dissolved in 0.05 ml N,N-dimethylformamide) in 1 ml 0.1 M citric acid phosphate buffer, pH 4.5 or 5. 0.02 ml hexazotized p-rosaniline or new fuchsine/ml or tetrazotized BAXD or 0.5 mg Fast Blue B or Garnet GBC/ml were employed as a coupling reagent. Hexazotized new fuchsine yields the best results independent on the pretreatment of the tissue and the organ investigated followed by hexazonium-p-rosaniline. Compared with the azo dye method using naphthol AS-BI beta-D-N-acetyl-glucosaminide as a substrate and hexazotized p-rosaniline or new fuchsine or tetrazotized BAXD for simultaneous coupling especially the azoindoxyl technique with the new fuchsine is equvialent or superior. When the indolyl glucosaminide is used in the indigogenic, tetrazolium or metal precipitation method the results are mostly inferior with the exception of the tetrazolium reaction using BSPT. However, the main advantage of the azoindoxyl procedure is that at least the azoindoxyl dye deriving from hexazotized p-rosaniline can be osmificated and withstands treatment with organic solvents and resins. Therefore, the reaction product seems to be suitable for the electron microscopic demonstration of glucosaminidase. Among the other reaction principles this can reliably be achieved only with BSPT as a tetrazolium salt followed by osmification of its formazan. After fixation of blocks of tissue in form- or glutaraldehyde beta-D-N-acetylglucosaminidase can be localized with 5-Br-3-indoxyl-beta-D-N-acetylglucosaminide as a substrate and hexazotized new fuchsine for simultaneous coupling in the lysosomes of many rat organs.

Acetylglucosaminidase↗

PubMed1979

Secretion of beta-N-acetylglucosaminidase isoenzymes by cultured cystic fibrosis fibroblasts.

Secretion of the lysosomal enzyme beta-N-acetylglucosaminidase (EC 3.2.1.30) by normal and cystic fibrosis fibroblasts has been compared. In contrast to an earlier report, no differences were found in either the rate of secretion, or in the molecular forms of the enzyme secreted by normal fibroblast cultures and cultures initiated from patients with cystic fibrosis. The B isoenzyme of beta-N-acetylglucosaminidase from cystic fibrosis fibroblasts was converted to a more negatively charged form during preincubation for 3 h at pH 5.0 and 37 degrees C. The enzyme from normal fibroblasts was unaffected by this treatment.

Acetylglucosaminidase↗

PubMed1975

Effects of ten steroids on acridine orange uptake and beta-N-acetylglucosaminidase levels in rat liver lysosomes.

Ten steroids have been compared for their ability to modify the rate of uptake of acridine orange by rat liver and by rat liver lysosomes in vivo. The short-term effects of the ten steroids on the specific activity of a lysosomal enzyme, beta-N-acetylglucosaminidase, were also compared. Five of the ten steroids were administered as tritium-labelled compounds and the concentration of steroids or metabolites was measured in rat liver and liver lysosomes at 2.5h and 3.75h after administration. Cortisone acetate, etiocholanolone (5-beta-androstan-3-alpha-01-17-one) and testosterone accelerate and increase the uptake of acridine orange by rat liver lysosomes. Deoxycorticosterone, corticosterone, triamcinolone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione), estradiol-17-beta and progesterone appear to inhibit the uptake of acridine orange by rat liver lysosomes at 2.5 hours. Cortisol and dexamethasone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione) had little effect. All steroids with the exception of etiocholanolone and deoxycorticosterone increase with the specific activity of beta-N-acetylglucosaminidase in the lysosomal fraction at 2.5h. None of the effects at 2.5h are due to lowered protein levels. Lysosomal concentrations of radioactivity following the administration of tritiated steroids were greated for the glucocorticoids, corticosterone and cortisol. Estradiol-17-beta, progesterone and testosterone showed much lower concentrations of radioactivity in isolated lysosomes. Most of the lysosomal radioactivity (73-96%) was associated with the soluble fraction of the disrupted lysosomes.

17-Ketosteroids↗

PubMed1979

Core tetrasaccharide liberated by endo-beta-D-N-acetylglucosaminidase D from lactosamine-type oligosaccharides of Semliki Forest virus membrane proteins.

[3H]Mannose- and [3H]glucosamine-labeled lactosamine-type glycopeptides of Semliki Forest virus membrane proteins were stripped of their fucose, sialic acid, galactose and distal N-acetylglucosamine residues and subsequently digested with endo-beta-D-N-acetylglucosaminidase D from Diplococcus pneumoniae. Two products were obtained, a neutral tetrasaccharide and a residual glycopeptide fraction. The tetrasaccharide appeared to consist of two alpha-mannose residues, one beta-mannose residue and one N-acetylglucosamine residue located at the reducing terminus of the molecule. Results of Smith degradation, beta-elimination and acetolysis were compatible with four structures; (1) Man alpha-1-3[Man alpha 1-6]Man beta 1-4GlcNAc; (2) Man alpha 1-3Man beta 1-4[Man alpha 1-6] GlcNAc; (3) Man alpha 1-3Man alpha 1-4[Man beta 1-6]GlcNAc, or (4) Man alpha 1-6Man alpha 1-3Man beta-1-4GlcNAc. The reactivity of the viral glycopeptides with endo-beta-D-N-acetylglucosaminidase D and the chromatographic properties of the liberated core tetrasaccharide suggest that its most likely structure was Man alpha 1-3[Man alpha-1-6]Man beta 1-4GlcNAc. The core tetrasaccharide of glycans of membrane protein E3, one of the viral membrane proteins obtained from infected cell, was similar to that of the virion glycans.

Acetylglucosaminidase↗

PubMed1978

Secretion of beta-N-acetylglucosaminidase isoenzymes by normal human fibroblasts.

1. Secretion of the lysosomal enzyme beta-N-acetylglucosaminidase (EC 3.2.1.30) by normal human fibroblast cultures was linear with respect to time up to 96h. 2. Two forms of the A isoenzyme of beta-N-acetylglucosaminidase were found in the culture medium. One form was similar to the isoenzyme found in other extracellular fluids, such as plasma and tears, the other resembled the intracellular (lysosomal) enzyme. The presence of the two isoenzymes in the culture medium appears to reflect two distinct secretory processes. 3. It is suggested that plasma acid hydrolases may be destined for incorporation into lysosomes in a manner analogous to that described for the packaging of lysosomal enzymes by fibroblasts.

Acetylglucosaminidase↗

PubMed1978

Genetic analysis of the gene for N-acetylglucosaminidase in Dictyostelium discoideum.

Three independent mutations affecting N-acetylglucosaminidase in Dictyostelium discoideum were mapped by the parasexual system and found to lie on linkage group IV. These mutations as well as two others were found to be recessive and noncomplementing in heterozygous diploids. Thus they all appear to affect the nagA locus. Since two of the mutations give rise to thermolabile enzyme, this defines the structural gene for N-acetylglucosaminidase. The enzyme is a homodimer of a 68,000 dalton subunit and thus would be expected to be determined by a single locus. The expression of this gene is regulated by the stages of development; however, it should be mentioned that none of the mutations fell in a separate locus that might determine a specific positive regulatory proteins.

Acetylglucosaminidase↗

PubMed1976

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.

Acetylglucosamine↗