Cleavage of the (1 goes to 3)-2-acetamido-2-deoxy-beta-D-glucopyranosyl linkage present in keratan sulfate. The A and B isoenzymes of human liver hexosaminidase (EC 3.2.1.30)

The disaccharide 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 goes to 3)-D-[1-3H]-galactitol, prepared from keratan sulfate, was rapidly hydrolyzed by the A and B isoenzymes of normal human liver hexosaminidase (EC 3.2.1.30), and by the B isoenzyme prepared from the liver of a patient who had died of Tay-Sachs disease. The disaccharide substrate was also hydrolyzed by extracts of normal, cultured-skin fibroblasts, and fibroblasts of patients with Tay-Sachs disease, whereas it was not hydrolyzed by fibroblast extracts of patients with Sandhoff disease. Thus, effective degradation of keratan sulfate, secondary to a defect of the beta subunits present in the A and B isoenzymes of hexosaminidase, may contribute to the appearance of skeletal lesions in patients affected by Sandhoff disease.     

Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the purified enzyme, Sephadex G-50 chromatography indicated the presence of a number of oligosaccharides differing in size and sulfate content. The characterization of two major fractions of the oligosaccharides indicated that the point of enzyme attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated D-galactose residues participate. The enzyme, unlike ordinary exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to 3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of this enzyme, suggesting that a structure of the type, X-(1 leads to 3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate molecule must have an important effect. Compared with corneal keratan sulfate, keratan sulfates from human nucleus pulposus and shark cartilage were attacked at lower rates with a resultant production of oligosaccharides of relatively large size. The result is in agreement with the view that considerable variations exist in the structure of keratan sulfates of different origin, and further suggests that the enzyme may serve as a useful reagent in studying these variations.     

Purification of apo-3-D-(-) hydroxybutyrate dehydrogenase from rat liver mitochondria

Summary 3-D-(-) hydroxybutyrate dehydrogenase (EC 1.1.1.30) from rat-liver mitochondria was purified in the form of the soluble, phospholipid-free apoenzyme by a procedure involving: (1) solubilization of the membrane bound enzyme by controlled digestion of membrane phospholipids with porcine pancreas phospholipase A₂; (2) stabilization and separation of the released apoenzyme as a complex with egg-lecithin by gel filtration on Sephadex G-100; and (3) specific displacement of the apoenzyme from the enzyme-lecithin complex by treatment withBothrops atrox venom phospholipase A₂ (in the absence of Ca²⁺ ions) and subsequent separation of the displaced apoenzyme by gel filtration on Sephadex G-100. The method described is adequate for samples containing about 40 mg of mitochondrial protein. The yield in activity is 42% of that present in mitochondria and the degree of purification of the apodehydrogenase is about 170 fold. The purified apodehydrogenase shows one single sharp band when submitted to SDS polyacrylamide gel electrophoresis, with a mobility corresponding to a molecular weight of 38000 daltons. Gel filtration of the apoenzyme on Sephadex G-100 shows two active peaks with molecular weights of 76000 and 38500 daltons, indicating two different states of aggregation, namely, monomer and dimer. The corresponding diffusion coefficients are 7.73 (monomer) and 5.70 (dimer) × 10^–7. The apodehydrogenase preparation is devoid of phospholipids and is catalytically inactive. It can be reactivated by addition of egg lecithin or phospholipid mixtures containing lecithin in a suitable physical state. Reactivation occurs after formation of an active apodehydrogenase phospholipid complex.Abbreviations HBD 3-D-(-) hydroxybutyrate dehydrogenase - apoHBD 3-D-(-) hydroxybutyrate dehydrogenase apoenzyme - SMP submitochondrial particles - DFP diisopropylfluorophosphate - BSA bovine serum albumin - MPL mitochondrial phospholipids - L-diC₁₄ 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine - lysoC₁₄ 1-myristoyl-sn, glycero-3-phosphorylcholine - D-diC₁₀ 2.3-didecanoyl-sn-glycero-1-phosphorylcholine - tlc thin layer chromatography - SDS sodium dodecylsulfate Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Phenylalanyl-tRNA synthetase of wheat embryos. Purification, molecular weight, structure, properties]

From wheat germ, a phenylalanyl-tRNA synthetase (E.C.6.1.1.20) has been isolated and purified 187 fold by means of ammonium sulfate fractionation (40-50 per cent) followed by Sephadex G-200 gel filtration, chromatographies on DEAE-cellulose and hydroxyapatite. The enzyme appears to be homogeneous on Sephadex G-200 molecular filtration and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation, gel filtration and gel electrophoresis give an average of 250 00 daltons. The enzyme is dissociated in 1 per cent sodium dodecyl sulfate into two different equimolar components of 80 000 and 50 000 daltons ; this result suggests that the phenylalanyl-tRNA synthetase has a subunit structure : alpha2 beta2. Dissociation with sodium dodecyl sulfate and dithiothreitol gives four other components, probably resulting from the breakdown of the subunits. Optima values of pH, Mg2+ and K+ concentrations, effect of SH-compnents, kinetic parameters have been determined in the aminoacylation reaction. Physical and catalytic properties of wheat germ phenylalanyl-tRNA synthetase appear very similar to those of the yeast and E. coli enzymes.     

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues. n-Butyramide, 3-chloropropionamide, 3-aminopropionamide, and isovaleramide analogues

The syntheses of four analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. Activated carboxylic acids were reacted with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. n-Butyric anhydride gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-n-butyramide. 3-Chloropropionic anhydride was synthesized from 3-chloropropionic acid and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-chloropropionamide. Equilibration of the latter with ammonium bicarbonate gave N1-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-aminopropionamide. Succinimidyl isovalerate was synthesized and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-isovaleramide.     

Purification and characterization of glutathione reductase from bovine erythrocytes

Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid

Inhibin from bovine follicular fluid was partly purified using affinity chromatography on immobilized Procion Red 3B, gel filtration on Sephadex G-25 and ion-exchange chromatography on the fast protein liquid chromatography system. Inhibin was subsequently characterized using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. Biological activity was associated with a protein with an apparent molecular weight of approximately 65 kD.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

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.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Oxidation of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1-propene compounds and the structure of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1,2-propanediol derivatives for a synthesis of 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid

Oxidation of 5-acetamido-4,8-anhydro-1,2,3,5-tetradeoxy-D-glycero-D-ido-non-1-enitol [3-C-(2-amino-2-deoxy-beta-D-glucopyranosyl)-1-propene] was studied to search for preparative routes to aminodeoxy didehydro nonulosonic acid derivatives. Since only moderate chiral induction was observed with osmium tetroxide dihydroxylation as well as with peracid epoxidation, the catalytic asymmetric dihydroxylation conditions were applied to give the stereocontrolled formation of 1,2-propanediol derivatives. The structures of these diastereoisomeric 1,2-propanediol derivatives were determined by X-ray crystallographic analyses. The formation of diastereoisomeric 1,2-propanediols also varied with the nature of 2-substituent on the aminodoexy glycosyl moiety. Thus 5-acetamido-4,8-anhydro-3,5-dideoxy-D-erythro-L-ido-nonitol [(2S)-3-C-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-1,2-propanediol] was obtained predominantly up to 70% from 3-C-(2-acetamido-2-deoxyglycosyl)-1-propene by the use of ADmixbeta reagent. The (2S)-propanediol derivative was transformed in a five-step reaction sequence to 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid.     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

Production and purification of lipase by a mutant strain ofRhizopus arrhizus

Extracellular lipase production byRhizopus arrhizus was increased by mutant selection from 130 to 670 μmol FFA per mL per min using UV radiation and aziridine treatment. The produced lipase was purified 720-fold by ammonium sulfate fractionation and Sephadex G-100 gel filtration. The molar mass of the produced lipase was determined to be approximately 67 kDa which comigrated with bovine serum albumin in both a Sephadex G-100 column and SDS-PAGE.     

Isolation and characterization of an endo-beta-D-glucuronidase from the fungus Kobayasia nipponica

An endo-beta-D-glucuronidase was isolated and characterized from Kobayasia nipponica. The enzyme was purified by ammonium sulfate fractionation, CM-Sephadex chromatography, gel filtration with Sephacryl S-200, and heparin-Sepharose chromatography. The enzyme shows the following properties: optimum pH 5.0, thermal stability below 37 degrees C, pH stability 5-6, optimum temperature 45-55 degrees C, and Km 0.12% for L-idurono-D-glucuronan (protuberic acid (PA), L-IdUA:D-GlcUA = 1:2) from Kobayasia nipponica, 0.19% for PF (L-IdUA:D-GlcUA = 1:3) from Pseudocolus fusiformis, and 0.23% for (1-4)-beta-D-glucuronan(mucoric acid) from Mucor mucedo as determined from Hofstee plots. The molecular weight values estimated by gel filtration through Sephacryl S-200 and Sephadex G-50 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were 10,500 and 10,200, respectively. The endo-beta-D-glucuronidase was inactive towards several glycosaminoglycans.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.