Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Purification of H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase from human serum

The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.     

Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.

During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.     

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Chemical structure of neutral sugar chains isolated from human mature milk kappa-casein

The carbohydrate chains linked to human kappa-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and two hexasaccharide alditols (10.9%] were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal beta 1----3GalNAc-ol (I), Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (II), Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (III), GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (IV), GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (V), Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (VI) and Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (VII). Five oligosaccharide alditols (III-VII) were the novel carbohydrate chains of kappa-casein from mammalian milk.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Bowringia milbraedii agglutinin. Specificity of binding to early processing intermediates of asparagine-linked oligosaccharide and use as a marker of endoplasmic reticulum glycoproteins

We have purified a protein with hemagglutinating activity from the seeds of a West African legume, Bowringia milbraedii. The purified protein, designated BMA, has a native Mr = 38,000 on gel filtration and a subunit size of Mr = 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Hemagglutination was inhibited most effectively by Man alpha 1----2 linked sugars. Affinity chromatography of oligosaccharides on BMA-Sepharose showed that Man alpha 1----2Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol (where GlcNAcol is N-acetylglucosaminitol) and Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol were retarded on the column, whereas Man alpha 1----3Man beta 1----4GlcNAcol did not bind. Oligomannosidic-type glycans obtained by treatment of [3H] mannose-labeled baby hamster kidney cells with endo-beta-N-acetylglucosaminidase H bound more strongly to BMA-Sepharose and required 10 or 200 mM methyl-alpha-mannoside for elution. Oligosaccharides bearing the sequence Man alpha 1----2Man alpha 1----6Man alpha 1----6Man, i.e. Man9GlcNAc and certain isomers of Man8GlcNAc and Man7GlcNAc, bound more tightly than other Man8 GlcNAc and Man7GlcNAc isomers lacking this sequence. Man6GlcNAc and Man5GlcNAc were weakly bound. These results suggest that BMA binds preferentially to glycoproteins that are subjected to early steps of oligosaccharide processing in the endoplasmic reticulum but not to glycoproteins that are exposed to more extensive processing by Golgi mannosidases. Staining of permeabilized cells with BMA-chromophore conjugates revealed a reticular cytoplasmic pattern consistent with a preferential visualization of the endoplasmic reticulum. BMA staining was less evident in the juxtanuclear regions that were stained brightly with wheat germ agglutinin, a lectin that binds preferentially to sialylated glycoproteins located in Golgi compartments.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Isolation and some properties of a new enzyme-binding protein in rat plasma.

alpha-1-Inhibitor3 (alpha-I3), a new enzyme-binding protein, was isolated from rat plasma by a combination of ammonium sulfate precipitation, ion exchange chromatography on DEAE cellulose and gel filtration on ultrogel AcA34. Agarose gel electrophoresis of the purified inhibitor showed a single protein band with alpha1-mobility giving a single precipitation line on immunoelectrophoresis against anti-rat serum. A specific antiserum against the purified inhibitor was raised in rabbits. alpha1-I3 showed immunologic cross-reaction with human inter-alpha-trypsin inhibitor. alpha1-I3 formed a complex with trypsin, which was thereby inhibited; the electrophoretic mobility of the complex was less than that of free inhibitor. Inflammation, induced by turpentine, caused a decrease in the serum concentration of alpha1-I3 to 36% of the initial value within 48 h. alpha2 acute phase macroglobulin (alpha2-AP) showed a simultaneous increase to 7.1 g/l and alpha1-antitrypsin (alpha1-AT) to twice its normal value.     

Purification and properties of a coagulant proteinase isolated from bushmaster (Lachesis muta) venom (Serpentes: Viperidae)

The venom of Lachesis muta is a rich source of a thrombin-like enzyme. Its coagulant proteinase was purified by DEAE -Sephadex A -50 followed by agmatine CH -Sepharose and gel filtration on Sephadex G-100. On polyacrylamide gel electrophoresis at pH 8.4 a single band was observed. Its molecular weight by gel filtration was 49,000. The coagulant and esterolytic activities toward human fibrinogen and Tame of the inudasa were 662 NIH units/mg of protein and 4.37 delta OD225/min x 10(-3)/micrograms/ml, respectively. These values represent 23 and 5.7 fold increase over the crude venom. The enzyme mudasa, was evaluated with serum from human patients at Hospital Nacional de Ni?os Dr. Carlos Sáenz Herrera and found to be a valuable reagent for the quantification of fibrinogen on heparinized plasma.     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.     

Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.