Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus.

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.     

Purification and Properties of β-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced β-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher β-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had β-N-acetylgalactosaminidase activity equivalent to about 40% of its β-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for β-N-acetylglucosaminidase activity and 5.5 to 6.5 for β-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

Sheep liver beta-N-acetylhexosaminidase: partial purification, characterization and photodynamic inactivation

Sheep liver beta-N-acetylhexosaminidase was purified over 20-fold by conventional methods. The enzyme possessed activity against both p-nitrophenly-beta-D-N-acetylglucosaminide and p-nitrophenyl-beta-D-N-acetylgalactosaminide as substrates. On the basis of a variety of physical and chemical analyses including pH stability, substrate inhibition studies and photodynamic inactivation, it was concluded that both the beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside within the same molecule.     

Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller.

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8--4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 2. Kinetic studies on the active site

The purified beta-N-acetylglucosaminidase isolated from Turbatrix aceti hydrolyzes both p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides. The enzyme had Km values of 0.28 and 0.23 mM, Vmax values of 104 and 69 mumol min-1 mg protein-1, and activation energies of 11.7 and 9.9 kcal/mol for the two substrates, respectively. Several lines of experimental evidence show that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same molecule at a single catalytic site. Substrate analogs were synthesized in which the acetamido group of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside, and their 1-thio analogs was modified by replacement of the amido-carbonyl oxygen with sulfur. These substrate analogs competitively inhibited both enzymatic activities. Analysis of the inhibition data indicates that a single catalytic site of the enzyme is responsible for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Competition kinetics between the two substrates further confirm the presence of a single active site for both activities. The pH dependence of the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides has been determined. pKe1 and pKe2 values of 4.7 and 5.2, determined from the dependence of log Vmax/Km on pH, suggest that two carboxyl groups are involved in the reaction mechanism. The heats of ionization of the groups further confirm the above results.     

Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral beta-N-acetylhexosamindases

The bulk of rat brain neutral beta-N-acetylhexosaminidases (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) were present in the cytosol fraction. They were not bound by concanavalin A-Sepharose while the acid beta-N-acetylhexosaminidases were all bound. The neutral beta-N-acetylgalactosaminidase had a pH optimum of 5.2 and Km of 0.57 mM, while the neutral beta-N-acetylgalactosaminidase had the highest reaction rate at lost more than 90% of the activity in 30 min at 50 degrees C. The galactosaminidase pH 6.0 with a Km of 0.12 mM. No divalent ions activated either of the enzymes. The galactosaminidase was heat-stable and lost only 10--20% of its activity after 3 h at 50 degrees C. The neutral glucosaminidase was inhibited by free N-acetylglucosamine but not by N-acetylgalactosamine. The reverse was found for the neutral beta-galactosaminidase. Two enzymes were separated almost completely by hydroxyapatite chromatography. Heat stability of the separated activity peaks suggested that the neutral beta-N-acetylgalactosaminidase, which was not bound to hydroxyapatite, may be specific to the galactosaminide substrate. The neutral beta-N-acetylglucosaminidase may, on the other hand, have some activity toward the galactosaminide substrate. Both of the neutral enzyme activities were highest during the first postnatal week in rat brain in contrast to the acidic enzyme which showed peak activities during the second and third weeks. These results confirmed and expanded earlier observations by Frohwein and Gatt in calf brain. The relationship of these enzymes to the hexosaminidase C in human tissues is less certain at the present time.     

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon.

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.     

Immunological evidence to show that the N-acetylglucosaminidase and N-acetylgalactosaminidase activities of Dictyostelium discoideum reside in the same protein molecule (Short Communication)

Antibodies were produced against purified beta-N-acetylhexosaminidase from Dictyostelium discoideum. Ouchterlony tests produced a single precipitin band with both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Both activities were co-precipitated in exactly the same proportions in quantitative immunoprecipitation reactions. We conclude that one enzyme protein catalyses the hydrolysis of both N-acetylglucosaminides and N-acetylgalactosaminides.     

Immunological studies on CTP:phosphocholine cytidylyltransferase from the livers of normal and choline-deficient rats

Chickens were immunized with the purified low-molecular-weight form of CTP:phosphocholine cytidylyltransferase from rat liver cytosol. The antiserum was obtained and fractionated to yield immunoglobulin. The antibodies specifically inhibited the enzymatic activity of the partially purified low-molecular-weight form of the enzyme from pH 6.0 to 8.5. Antibodies against the low-molecular-weight form of the enzyme cross-reacted with the high-molecular-weight form of the enzyme from cytosol as well as with the cytidylyltransferase associated with the microsomal fraction. The antibodies were used for the immunochemical determination of the amount of cytosolic phosphocholine cytidylyltransferase in the livers of normal and choline-deficient rats. The amount of enzyme in rat liver cytosol was not changed for at least 18 days of choline deficiency. The decrease in specific activity of the enzyme in choline-deficiency may be caused by factors other than adaptive changes in the level of enzyme.     

Purification, characterization and kinetics of beta-N-acetylhexosaminidases A and B from the slug Arion rufus L

Two beta-N-acetylhexosaminidases have been purified to homogeneity and characterized, from the digestive gland of the slug A. rufus L., showing very high specific activities. Hexosaminidase A (Hex A) was purified 1300-fold with a yield of 12%, and hexosaminidase B (Hex B) was purified 1400-fold with a yield of 20%. Purified Hex A or Hex B run as a single protein band in polyacrylamide gel disc electrophoresis, showing different mobilities. The purified preparations do not show any of the other glycosidase activities present in the crude extract. beta-N-acetylglucosaminidase (GlcNAc-ase) and beta-N-acetylgalactosaminidase (GalNAc-ase) activities are always associated in a single peak for each enzyme form, with constant activity ratio, in all the purification steps, since they are catalyzed by the same enzyme (Hex A or Hex B). The optimal pH for both forms are 4.5 for GlcNAc-ase and 4.0 for GalNAc-ase activity. Hex B shows thermal and pH-stability higher than Hex A. The isoelectric points are 4.5 and 5.5 for A and B forms, respectively. The molecular weight is 150 000 for Hex A and 320 000 for Hex B. The amino acid composition of purified Hex A and B presents some differences concerning particularly Cys, Thr, Ser, Glu and Ile. The ratios Vmax/Km show that GlcNAc-ase is the main activity of both enzyme forms. beta-N-acetylglucosides and beta-N-acetylgalactosides completely compete for a common active site in mixed-substrates experiments. The Ki values are always coincident for GlcNAc-ase and GalNAc-ase activities, using competitive inhibitors (the corresponding lactones). These results strongly suggest that both activities are catalyzed by the same active site in both Hex A and B. Inhibition of the enzyme activities was found with the corresponding lactones, N-acetyl hexosamines, mannose, mannosides, HgCl2 and lead acetate; activation, with ribose, and with some chlorides and sulphates of divalent cations.     

Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms.

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.     

Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.     

Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography.

A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].     

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.     

Partial purification and properties of a β-N-acetylglucosaminidase from the fungus Sclerotinia fructigena

1. As cultures of the fungus Sclerotinia fructigena autolysed, the filtrates contained increasing quantities of a beta-N-acetylglucosaminidase. 2. The enzyme was purified up to 42-fold by a combination of isoelectric focusing and gel filtration. 3. It ran as a single band in cellulose acetate strip electrophoresis and in isoelectric focusing (pI3.76). 4. The enzyme did not readily hydrolyse chitin or a glycopeptide with terminal N-acetylglucosamine residues, but rapidly degraded the N-acetylglucosamine dimer NN'-diacetylchitobiose; the monomer was readily utilized by the fungus as a nitrogen source. The K(m) value for hydrolysis of p-nitrophenyl beta-2-acetamido-2-deoxy-d-glucopyranoside at 37 degrees C was 2.0mm. The Sclerotinia enzyme was generally less susceptible to inhibition by 2-acetamido-2-deoxygluconolactone and other related sugars than the corresponding enzyme from other sources. Inhibition by excess of substrate was observed. 5. The culture filtrate also contained N-acetylgalactosaminidase activity; conflicting evidence was obtained as to whether the same enzyme was responsible for both hexosaminidase activities.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Rates of degradation and synthesis of glycosidases de novo during growth and differentiation of Dictyostelium discoideum.

1. Injection of a purified preparation of beta-N-acetylglucosaminidase from the spent growth medium of myxamoebae of Dictyostelium discoideum into rabbits gave rise to an antibody preparation containing both anti-alpha-glucosidase and anti-beta-acetylglucosaminidase activities. 2. These two activities were shown to reside in different immunoglobulin molecules and it was concluded that the beta-N-acetylglucosaminidase preparation contained trace amounts of highly antigenic alpha-glucosidase. 3. A single precipitin band having beta-N-acetylglucosaminidase activity was formed in Ouchterlony plates when this antibody preparation was tested against extracts obtained from differentiated cells or from myxamoebae grown either axenically or on bacteria. 4. The antibody preparation was used to show that both beta-N-acetylglucosaminidase and alpha-glucosidase molecules are synthesized de novo from isotopically labelled amino acids during both the growth and differentiation phases of the life cycle and to show that neither of these proteins is significantly degraded during the growth phase or during the first 9h of differentiation. 5. The rates of accumulation of these assayable enzyme activities are thus equal to their rates of synthesis during growth and early differentiation. 6. The factors regulating cellular enzyme activity during the life cycle of D. discoideum are discussed.     

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.     

Purification and Properties of an Amylase from Bacillus cereus NY-14

An extracellular amylase was obtained from a culture medium of Bacillus cereus NY-14. This enzyme was purified to show a single band on disc gel electrophoresis by ammonium sulfate fractionation and Sephadex G-100 gel filtration to 1101-fold of the activity of the original culture liquor. The purified enzyme had a molecular weight of 55,000, an isoelectric point of 6.13, an optimum pH of 6.0, and an optimum temperature of 55°C. The pH-stability range was wide; the enzyme retained more than 80% of its initial activity in the range of pH 5.5 to 12. It was stable below 35°C and required calcium ions for the stabilization. The action pattern of this enzyme on amylaceous polysaccharides was unique in that the predominant product was maltopentaose. The purified amylase could also digest starch granules of such plants as rice, barley, corn, and kuzu to produce maltopentaose as the main product.     

Cell-specific beta-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton.

It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a variety of compounds within the dissolved organic matter (DOM) pool in marine environments. Herein, cultures and field populations of marine phytoplankton were assayed for beta-N-acetylglucosaminidase activity, a terminal enzyme of chitin degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not cell-specific, was complemented with a cell-specific assay that images the activity associated with single cells using an enzyme labeled fluorescence (ELF) substrate. beta-N-acetylglucosaminidase activity was widespread across various taxa of marine phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied with the nutritional physiology of the test species in three of the 17 cultures tested. In these three cases the number of cells with activity in the low nutrient medium was higher than in nutrient replete medium. Taken together, these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-like DOM degradation and should be incorporated into conceptual models of chitin cycling in marine systems.