Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Interaction of a rat intestinal brush border membrane glycoprotein with type-1 fimbriae of Salmonella typhimurium

Type-1 fimbriated Salmonella typhimurium was found to adhere to rat intestinal brush border membrane in a mannose sensitive manner. The maximum binding of the purified fimbriae observed with the rat illeal enterocytes was inhibited by 69.2% in presence of D-mannose. Brush border membrane from rat illeum was isolated, delipidified, solubilised and fractionated by affinity chromatography on type-1 fimbriae coupled Sepharose CL 4B column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the material eluted from the column with D-mannose revealed a single band of molecular weight 60 kDa. The direct binding of this affinity eluted glycoprotein to the purified type-1 fimbriae was demonstrated by a modified Western blot experiment. Our findings suggest that the 60 kDa glycoprotein may serve as a receptor for the type-1 fimbriae in the rat intestinal brush border membrane and thereby may help in mediating bacterial adherence to the host epithelium.     

A novel protein accumulated during maturation of the pods of the plantImpatiens balsamina

A high molecular weight protein has been isolated as a major polypeptide comprising 85% of the total extractable proteins in the fruit pericarp of the plantImpatiens balsamina. The protein has been purified to homogeneity following fractionation of the crude cell supernatant with ammonium sulphate, chromatography on Sepharose 4B and Sephacryl-S-300. This protein appeared to be a homo-tetramer consisting of subunits, Mr 75 K. Amino acid analysis showed the presence of more acidic amino acids, with an isoelectric point 5.8. The interaction of this protein with filamentous actin, both from rabbit muscle and Mung bean embryo was monitored by transmission electron microscopy, light scattering analysis, viscometry and sedimentation assay. This protein inhibited appreciably the polymerization of G-actin to F-actin.     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production

Fungi were isolated from natural soil samples and screened for extracellular dextranase synthesis. The strain F1002 was identified as Hypocrea lixii using a standard internal transcribed spacer ribosomal DNA analysis and was selected for extracellular dextranase synthesis. The enzyme was purified via ammonium sulfate precipitation and Sepharose 6B chromatography, which resulted in an 8.3-fold increase in the specific activity and a 10.73% recovery. This enzyme is a monomeric protein with a molecular mass of 62 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme, which was identified as an endodextranase, had an optimum pH of 5.0 and an optimum temperature of 25 °C. The dextranase activity was enhanced by Mg²⁺, Al³⁺, and especially Zn²⁺ at a low concentration, which improved its activity to 124.22%. The enzyme has a very high hydrolytic affinity toward high-molecular weight dextrans. Setting the concentrations of the H. lixii F1002 dextranase (2.31 U/mL) and dextrans (6%), as well as the reaction time (45 min), allowed the dextranase to hydrolyze dextrans of controlled molecular weights (20–70 kDa). Three types of oligodextrans with different molecular weights (namely, 69,376, 38,251, and 21,364 Da) were obtained, with a total yield of 80.32%.     

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Summary Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent K _m value was 0.164 mg ml⁻¹. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.     

Effects of estradiol-17β in the male xenopus laevis: Isolation and translation of cytoplasmic messenger rna populations

Summary Total Xenopus liver cytoplasmic RNA isolated following long-term estrogen administration (14 days) was fractioned using Sepharose 4B chromatography. One of the Sepharose 4B peaks was shown to contain RNA with a molecular weight reported for vitellogenin mRNA (34S). The presence of estrogeninduced vitellogenin mRNA in the peak 5 RNA was determined by translation of the RNA in the oocyte and analysis of the oocyte translational products by immunoprecipitation with anti-vitellogenin.Sepharose 4B peaks 2 and 3 were also observed to contain estrogen induced mRNA populations sedimenting between 9-18S. These findings suggest that Sepharose 4B chromatography might prove useful in separating different mRNA populations following estrogen-induced gene activation.     

Isolation and properties of a sialidase fromTrypanosoma rangeli

In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-(2–3)-lactose> N-glycoloylneuraminy-(2–3)-lactose> N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg²⁺ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca²⁺ Mn²⁺ or Mg²⁺ ions.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - CMP cytidine monophosphate - EDIA ethylenediaminetetraacetic acid - ESM equine submandibular gland mucin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - Lac lactose - MU-Neu5Ac 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4Ac5Gc N-glycoloyl-4-O-acetylneuraminic acid - Neu2en 2-deoxy-2,3-didehydroneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - PMSF phenylmethylsulfonyl fluoride - PSM pig submandibular gland mucin - SDS sodium dodecyl sulfate - Tris tris-(hydroxymethyl)aminomethane Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic componenets. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103 000 daltons. Xan VIa was a glycoprotein of molecular weight 17 000 daltons. The carbohydrate moiety of Xan Vla was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).     

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg²⁺ and Mn²⁺. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum

The F₄₂₀-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent K_m of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH₂ nor FADH₂ could serve as electron donors; NAD was not utilized as electron acceptor.     

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive     

Endothelial heparan sulphate: Compositional analysis and comparison of chains from different proteoglycan populations

From cultures of human umbilical vein endothelial cells incubated with³H-glucosamine or³⁵S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG heparan sulphate proteoglycan - DSPG dermatan sulphate proteoglycan - GlcNAc(6S) N-acetylglucosamine 6-sulphate - GlcNAc6R glucosamine with either-OH or-OSO₃ at C-6 - GlcNR glucosamine with either-SO₃ or-COCH₃ as N-substituent - GlcNSO₃ N-sulphated glucosamine - GlcNSO₃(3S) N-sulphated glucosamine 3-sulphate - GlcA d-glucuronic acid - IdoA l-iduronic acid - IdoA(2S) iduronic acid 2-sulphate - HexA hexuronic acid - DHexA hexuronic acid with a 4,5-double bond - Xyl xylose - SAX strong anion exchange - d.p. degree of polymerization (a disaccharide has d.p.=1 etc) - AUFS absorbance units full scale The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6].     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis