Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]     

Polar glycosphingolipids in insect: chemical structures of glycosphingolipid series containing 2'-aminoethylphosphoryl-(----6)-N-acetylglucosamine as a polar group from larvae of the green-bottle fly, Lucilia caesar.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.     

Purification and characterization of the epitectin from human laryngeal carcinoma cells

The purification and partial characterization of epitectin (previously called Ca antigen) from a human cancer cell line is described. This glycoprotein, which is expressed on a wide range of human tumors and certain specialized normal epithelia, can be detected using monoclonal antibodies, Ca1, Ca2, and Ca3. The purified glycoprotein had a high density (1.40 g/ml) on isopycnic centrifugation indicating a high carbohydrate content. The molecular mass of epitectin as determined by size-exclusion chromatography ranged from 1.0 to 1.5 x 10(6) daltons. However, the purified epitectin gave two bands of apparent molecular weight 390,000 and 350,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of epitectin and asialoepitectin were found to be 5.3-5.4 and 6.8, respectively. The oligosaccharides were isolated from metabolically labeled epitectin by alkaline borohydride treatment and their structures established based on high performance liquid chromatography and paper electrophoretic migration, sugar composition, the results of sequential exoglycosidase treatment, periodate oxidation, and methylation analysis. The structures of the three major fractions, which together account for about 80% of the radioactivity, were assigned as NeuNAc alpha 2----3Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----3Gal beta 1----3GalNAc(OH), and Gal beta 1----3 GalNAc(OH). The structures of the minor fractions were tentatively assigned as NeuNAc----Gal(NeuNAc----Gal----GlcNAc)----GalNAc(OH), Gal beta 1----(NeuNAc alpha 2----6)3GalNAc(OH), NeuNAc alpha 2----6GalNAc(OH), and GalNAc(OH). It is proposed that the protein sequence and/or the distribution of the saccharides on the protein core are the determinants on epitectin that are recognized by the Ca antibodies.     

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.     

Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1 sulfate and/or 1 phosphate residue (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). The other seven compounds, which represent approximately 60% of the isolated linkage hexasaccharides, were analyzed by chondroitinase ACII digestion in conjunction with high performance liquid chromatography and by 500-MHz one- and two dimensional 1H NMR spectroscopy. All seven compounds have the following conventional structure in common. [formula: see text] Two disulfated compounds have an O-sulfate on C-6 of the Gal-2 residue attached to xylitol in combination with an O-sulfate on C-4 or on C-6 of the GalNAc residue. The third disulfated compound has O-sulfate on C-6 of Gal-2, and also on C-6 of Gal-3. Two of the trisulfated compounds also have O-sulfate on C-6 of both Gal-2 and Gal-3 with in addition sulfate on C-6 or C-4 of GalNAc. The other two trisulfated compounds have O-sulfate on C-6 of Gal-2 and on C-4 of Gal-3 in conjunction with sulfate on C-6 or C-4 of GalNAc.     

Occurrence of GalNAcbeta1-4GlcNAc unit in N-glycan of royal jelly glycoprotein

Elsewhere, we characterized the structure of twelve N-glycans purified from royal jelly glycoproteins (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000)). Structural analysis showed that the typical high-mannose type structure (Man9-4GlcNAc2) accounts for about 72% of total N-glycans, a biantennary-type structure (GlcNAc2Man3GlcNAc2) about 8%, and a hybrid-type structure (GlcNAc1Man4GlcNAc2) about 3%. During structural analysis of minor N-glycans of royal jelly glycoproteins, we found that one had an N-acetyl-galactosaminyl residue at the non reducing end; most of such residues have been found in N-glycans of mammalian glycoproteins. By exoglycosidase digestion, methylation analysis, ion-spray (IS)-MS analysis, and 1H NMR spectroscopy, we identified the structure of the N-glycan containing GalNAc as; GlcNAc(beta)1-2Man(alpha)1-6(GalNAcbeta1 - 4GIcNAcbeta1 - 2Man(alpha)1 - 3)Manbeta1 - 4GlcNAc(beta)1-4GlcNAc. This result suggested that a beta1-4 GalNAc transferase is present in hypopharyngeal and mandibular glands of honeybees.     

Isolation and characterization of major urinary oligosaccharides excreted by a patient with type 3 GM1 gangliosidosis

Two major oligosaccharides were isolated from the urine of a patient with type 3 GM1 gangliosidosis. From structural studies including compositional sugar analysis, fast-atom bombardment mass spectrometry, direct-inlet chemical ionization mass spectrometry, methylation analysis, chromium trioxide oxidation, and proton magnetic resonance spectroscopy, their structures were deduced to be as follows: [formula: see text] Both oligosaccharides have beta-linked galactose at the non-reducing ends. Oligosaccharide 1 is one of the most common urinary oligosaccharides found in type 1 and type 2 GM1 gangliosidosis. Oligosaccharide 2, lacto-N-difucohexaose II, has not been described in the urine of GM1 gangliosidosis patients. Excretion of oligosaccharide 1 in the type 3 patient was much less than that of a type 2 patient. Thin-layer chromatographic analysis revealed that the excretion of oligosaccharides with higher molecular weight than that of oligosaccharide 1 (octasaccharide) in the type 3 patient was much less than that of a type 2 patient, raising the possibility that the mutant beta-galactosidase of type 3 GM1 gangliosidosis can still act to some extent on higher molecular weight oligosaccharides containing beta-linked galactose at the non-reducing end.     

Structural analysis of O-glycosidic type of sialyloligosaccharide-alditols derived from urinary glycopeptides of a sialidosis patient

Sialidosis urine was fractionated by gel filtration on Bio-Gel P-6. All pooled fractions containing carbohydrates showed the presence of small amounts of GalNAc in non-reducing position, besides free N-acetyllactosamine type of oligosaccharides as major constituents. The fractions were subjected to reductive alkaline borohydride degradation, after which the major part of GalNAc was recovered as N-acetyl-D-galactosaminitol (GalNAc-ol). The GalNAc-ol-containing material was separated from the N-glycosidic oligosaccharides by a second gel-filtration step on AcA 202. Subsequently, the O-glycosidic sialyloligosaccharide-alditols were subfractionated by anion-exchange chromatography on Mono Q. Structural analysis by 500-MHz 1H-NMR spectroscopy revealed two major components in all fractions, namely: NeuAc alpha 2-3Gal beta 1-3GalNAc-ol and NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc-ol. Furthermore, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6]GalNAc-ol was found as a minor component in some of the fractions. The presence of these carbohydrate chains in Bio-Gel fractions differing in molecular mass suggested that they are derived from glycopeptides which are heterogeneous in their peptide part.     

Isolation and characterization of a novel chondroitin sulfate from squid liver integument rich in N-acetylgalactosamine(4,6-disulfate) and glucuronate(3-sulfate) residues

Novel chondroitin sulfate (CS) chains with an average molecular mass of 79.6 kDa were purified from squid liver integument. A compositional analysis of the CS chains using chondroitinases (CSases) ABC and AC-I revealed a range of variably sulfated disaccharides with GlcAβ1→3GalNAc(6-sulfate), GlcAβ1→3GalNAc(4-sulfate), and GlcAβ1→3GalNAc(4,6-disulfate) as the major ones, significant amounts of rare 3-sulfated GlcA-containing disaccharides, and a small amount of nonsulfated GlcAβ1→3GalNAc. The CS chains exhibited neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons, which was abolished completely by digestion with CSase ABC or AC-I. Consequently, whether these CS chains interact with heparin-binding growth factors was tested in a BIAcore system. All of the growth factors exhibited concentration-dependent and specific binding. CS chains from squid liver integument, with their unique composition and strong biological activities, may be a good candidate for therapeutic application.     

Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.     

Further studies on the gangliosidic nature of the cholinergic-specific antigen, Chol-1

The antigen designated as Chol-1 beta, detected by an antiserum specific for cholinergic neurons, has been purified to homogeneity from ganglioside mixtures extracted from Torpedo electric organ and pig brain. The final products from the two sources behaved identically in a wide range of tests and gave coincident immunopositive and Ehrlich-positive spots after thin layer chromatography in seven different solvent systems; they were thus considered to be identical and to constitute a single, pure chemical species. Gas-chromatographic analysis revealed the presence of long-chain bases, glucose, galactose, N-acetylgalactosamine, and sialic acid in integral molar ratios of 1:1:2:1:3; the compound's reactivity to cholera toxin after Vibrio cholerae sialidase treatment on thin layer chromatography and the recovery of GM1 as sole product of exhaustive sialidase treatment identified it as a member of the gangliotetrahexosyl series. From the products of partial enzymatic desialylation and treatment with beta-galactosidase and a comparison of the compound's immunoreactivity to anti-Chol-1 antisera with that of other trisialogangliosides of defined molecular structure, we were able to assign a disialosyl residue alpha-Neu5Ac-(2----8)-alpha-Neu5Ac-(2----3)- to the inner galactose, and we suggest GalNAc as a possible site of linkage of the third sialic acid.     

Purification of human midcycle cervical mucin and characterization of its oligosaccharides with respect to size, composition, and microheterogeneity

Human cervical mucin was solubilized from the gel phase of pooled midcycle cervical mucus using 6 M guanidine hydrochloride and 10 mM dithiothreitol and was then alkylated with iodoacetamide. Mucin was then purified by gel filtration on Bio-Gel A-50m resin in buffer containing 0.1% sodium dodecyl sulfate. The purified mucin gave a single band upon electrophoresis in either 5% acrylamide or 1% agarose gels. Protein comprised 21% of the glycoprotein by weight and amino acid analysis revealed a high content of Ser and Thr. Saccharide analysis yielded approximate molar ratios of Fuc:Gal:GlcNAc:GalNAc:NeuAc = 1:2:1:1:0.5. Inorganic sulfate, 1% by weight, was detected, but mannose was absent. Reductive alkali treatment of mucin resulted in release of oligosaccharides with concomitant conversion of 77% of GalNAc to its reduced derivative N-acetylgalactosaminitol (GalNAcol) thus demonstrating O-glycosidic linkage of GalNAc to protein. Reduced oligosaccharides were purified by ion exchange chromatography on DEAE-cellulose, paper chromatography, and high resolution gel filtration on Bio-Gel P-2 resin. A total of 16 reduced oligosaccharides were identified by thin layer chromatography. These included neutral, sialylated, and sulfated oligosaccharides and they varied in size from a disaccharide to a nonasaccharide. The major neutral oligosaccharide isolated (21% of recovered GalNAcol) was a tetrasaccharide, Gal:GlcNAc:GalNAcol = 2:1:1, and the major acidic oligosaccharide isolated (11% of recovered GalNAcol) was a trisaccharide, Gal:GalNAcol:NeuAc = 1:1:1.     

Molecular weight as a factor in the excretion of monoquaternary ammonium cations in the bile of the rat, rabbit and guinea pig

1. The excretion in the bile and urine of intraperitoneally injected (14)C-labelled monoquaternary ammonium or pyridinium cations was measured in bile-duct-cannulated rats (ten compounds) and in guinea pigs and rabbits (six compounds). 2. Seven of these, namely N-methylpyridinium, tetraethylammonium, trimethylphenylammonium, diethylmethylphenylammonium, methylphenyldipropylammonium, dibenzyldimethylammonium and tribenzylmethylammonium, were excreted largely unchanged in the bile and urine. 3. 3-Hydroxyphenyltrimethylammonium, 3-bromo-N-methylpyridinium and cetyltrimethylammonium were metabolized to an appreciable extent in the rat. 4. In intact rats intraperitoneally injected trimethylphenylammonium (mol.wt. 136) was excreted mainly in the urine, dibenzyldimethylammonium (mol.wt. 226) was excreted in roughly equal amounts in the urine and faeces, and tribenzylmethylammonium (mol.wt. 302) was excreted mainly in the faeces. The faecal excretion of these compounds corresponded to their biliary excretion in bile-duct-cannulated rats. About 3-4% of tribenzyl[(14)C]methylammonium was eliminated as (14)CO(2). 5. In rats the extent of biliary excretion of four cations with molecular weights in the range 94-164 was less than 10% of the dose, whereas that of five cations with molecular weights 173-302 was greater than 10%. These results and other data from the literature suggested that the molecular weight needed for the biliary excretion of such cations to an extent of 10% or more of the dose was about 200+/-50. Studies with six cations in guinea pigs and rabbits suggest that this value applies also to these species. 6. The results suggest that the threshold molecular weight for the appreciable (>10%) biliary excretion of monoquaternary cations is different from that for anions (Millburn et al., 1967a; Hirom et al., 1972b). With rats, guinea pigs and rabbits, no significant species difference was noted, whereas with anions there is a marked species difference.     

Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins

A novel Mr 28,000 erythrocyte transmembrane protein was recently purified and found to exist in two forms, "28kDa" and "gly28kDa," the latter containing N-linked carbohydrate (Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642). Although 28kDa protein resembles the Rh polypeptides biochemically, structural homologies were not identified by immunoblot or two-dimensional iodopeptide maps. The NH2-terminal amino acid sequence for the first 35 residues of purified 28kDa protein is 37% identical to the 26-kDa major intrinsic protein of lens (Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. Cell 39, 49-59). Antisera to a synthetic peptide corresponding to the NH2-terminus of 28kDa protein gave a single reaction of molecular mass 28kDa on immunoblots of erythrocyte membranes. Selective digestions of intact erythrocytes and inside-out membrane vesicles with carboxypeptidase Y indicated the existence of a 5-kDa COOH-terminal cytoplasmic domain. Multiple studies indicated that 28kDa and gly28kDa proteins exist together as a multisubunit oligomer: 1) similar partial solubilizations in Triton X-100; 2) co-purification during ion exchange and lectin affinity chromatography; 3) cross-linking in low concentrations of glutaraldehyde; and 4) physical analyses of purified proteins and solubilized membranes in 1% (v/v) Triton X-100 showed 28kDa and gly28kDa proteins behave as a large single unit with Stokes radius of 61 A and sedimentation coefficient of 5.7 S. These studies indicate that the 28kDa and gly28kDa proteins are distinct from the Rh polypeptides and exist as a multisubunit oligomer. The 28kDa protein has NH2-terminal amino acid sequence homology and membrane organization similar to major intrinsic protein and other members of a newly recognized family of transmembrane channel proteins.     

Structural determination of oligosaccharides derived from lipooligosaccharide of Neisseria gonorrhoeae F62 by chemical, enzymatic, and two-dimensional NMR methods

F62 LOS of Neisseria gonorrhoeae consists of two major LOS components; the higher and smaller molecular weight (MW) components were recognized by MAbs 1-1-M and 3F11 respectively. Base-line separation of the two major oligosaccharide (OS) components from F62 LOS was achieved by Bio-Gel P-4 chromatography after dephosphorylation of the OS mixture. The structures of the two major OSs were studied by chemical, enzymatic, and 2D NMR methods [double quantum filtered COSY (DQF-COSY), delayed COSY (D-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), pure-absorption 2D NOE NMR] as well as methylation followed by GC/MS analysis. The OS component derived from the MAb 1-1-M defined LOS component was determined to have a V3-(beta-N-acetylgalactosaminyl)neolactotetraose structure (GalNAc is beta 1----3-linked to a neolactotetraose) at one of its nonreducing termini as shown below. The above pentaose is linked to a branched diheptose-KDO core in which a GlcNAc is alpha-linked. The OS component derived from the MAb 3F11 defined LOS component did not have a GalNAc residue. The rest of its structure was identical to that of the OS-1, and a neolactotetraose is exposed at its nonreducing terminus. [formula: see text]     

Purification and properties of a diheme cytochrome b561 of the Escherichia coli respiratory chain

A new b-type cytochrome, cytochrome b561 (Murakami, H., Kita, K., Oya, H., and Anraku, Y. (1984) Mol. Gen. Genet. 196, 1-5) was purified to near homogeneity from the cytochrome b561-amplified Escherichia coli K12 strain HM204/pAM5029. The purified cytochrome b561 was a single polypeptide with a molecular weight of 18,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point was determined to be 9.6. The difference spectrum of the cytochrome at 77 K shows a major alpha-absorption peak at 561 nm and a minor peak at 555 nm. The absolute spectrum at room temperature of the oxidized form of the cytochrome had an absorption peak at 414 nm, and that of the reduced form had peaks at 562, 530, and 428 nm. The oxidation-reduction potential of the cytochrome was estimated to be +20 mV. The cytochrome contained 91.2 nmol of heme/mg of protein, showing that it was a cytoplasmic membrane-bound, b-type diheme cytochrome.     

Mucin biosynthesis: purification and characterization of a mucin beta 6N-acetylglucosaminyltransferase.

We have purified, to apparent homogeneity, a mucin beta 6N-acetylglucosaminyltransferase (beta 6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Gal beta 1-3GalNAc alpha benzyl (Bzl) to stabilize the beta 6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Superflow affinity column equilibrated with 1 mM Gal beta 1-3GalNAc alpha Bzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 mumol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the beta 6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The Km values for UDP-GlcNAc and Gal beta 1-3GalNAc alpha Bzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified beta 6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAc beta 1-3Gal beta R as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a beta 1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all beta 6GlcNAc structures found in mucin-type oligosaccharides.     

Isolation and characterization of protein A24, a "histone-like" non-histone chromosomal protein.

In earlier studies, the nucleolar levels of protein A24 were found to be markedly decreased in the nucleolar hypertrophy induced by thioacetamide or during liver regeneration (Ballal, N.R., Goldknopf, I.L., Goldberg, D.A., and Busch, H. (1974) Life Sci. 14, 1835-1845; Ballal, N.R., Kang, Y.-J., Olson, M.O.-J., and Busch, H.J. Biol. Chem. 250, 5921-5925). To determine the role of protein A24, methods were developed for its isolation in highly purified form. Milligram quantities of highly purified protein A24 were isolated from the 0.4 N H2SO4-soluble proteins of calf thymus chromatin by exclusion chromatography on Sephadex G-100, followed by preparative polyacrylamide gel electrophoresis. Protein A24 was highly purified as shown by its migration as a single spot on two-dimensional polyacrylamide gel electrophoresis, its single NH2-terminal amino acid, methionine, and the production of approximately 50 peptides by tryptic digestion. Like histones 2A, 2B, 3, and 4. A24 was extractable from chromatin with 0.4 N H2SO4 or 3 M NaCl/7 M urea, but unlike most non-histone proteins or histone 1, protein A24 was not extracted with 0.35 M NaCl, 0.5 M HClO4, or 0.6 M NaCl. Protein A24 was present in only 1.9% of the total amount of histones 2A, 2B, 3 and 4; its molecular weight is 27,000.     

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.