Abstract  The Fe(0) species Fe(N2)(dmpe)2 exists in equilibrium with the previously unreported dimer, [Fe(dmpe2)2(μ-N2)]. For the first time these complexes, alongside Fe(N2)(depe)2, are shown unambiguously to produce N2H4 and/or NH3 upon addition of triflic acid; for Fe(N2)(depe)2 this represents one of the highest electron conversion efficiencies for Fe complexes to date.

Abstract  Fluorine-18 labeled (2S,4S)-4-fluoro-l-proline (cis-[18F]4-FPro) has been reported to be a potential positron emission tomography tracer to study abnormal collagen synthesis occurring in pulmonary fibrosis, osteosarcomas, mammary and colon carcinomas. In this paper, we report the stereospecific radiofluorination of (2S,4R)-N-tert-butoxycarbonyl-4-(p-toluenesulfonyloxy) proline methyl ester (at 110 degrees C) to produce diastereomerically pure cis-[18F]4-FPro in 38% radiochemical yield at the end of a 90-min synthesis. Investigation of the effect of temperature on the stereospecificity of nucleophilic fluorination showed that diasteriomerically pure cis-[18F]4-FPro or trans-[18F]4-FPro was produced at lower temperatures (85 degrees C110 degrees C) during the fluorination of (2S,4R) or (2S,4S) precursors, respectively. However, at higher temperatures (130 degrees C145 degrees C), fluorination of (2S,4R) precursor produced a mixture of cis-[18F]4-FPro and trans-[18F]4-FPro diastereomers with cis-[18F]4-FPro as the predominant isomer. Hydrolysis of the purified fluorinated intermediate was carried out either in one step, using 2 m triflic acid at 145 degrees C for 10 min, or in two steps where the intermediate was heated in 1 m HCl at 110 degrees C for 10 min followed by stirring at room temperature in 1 N NaOH for 5 min. The aqueous hydrolysis mixture was loaded onto an anion exchange column (acetate form for one-step hydrolysis) or an ion retardation column (two-step hydrolysis) followed by a C18 Sep-Pak (R) (Waters Corporation, Milford, MA, USA). Pure cis-[18F]4-FPro was then eluted with sterile water. We also report that epimerization of cis-[18F]4-FPro occurs during the two-step hydrolysis (H+ followed by OH-) of the intermediate, resulting in 5 +/- 3% trans-[18F]4-FPro, whereas the one-step acid hydrolysis yielded pure cis-[18F]4-FPro in the final product. Copyright (C) 2011 John Wiley & Sons, Ltd.

Abstract  A dithiolate-bridged Fe-Ni-Fe trinuclear carbonyl complex [(CO)(3)Fe(mu-ndt)Ni(mu-ndt)Fe(CO)(3)] (1, ndt = norbornane-exo-2,3-dithiolate) has been synthesized from the reaction of [Fe(CO)(4)I(2)] and Li(2)[Ni(ndt)(2)]. This reaction was found to occur with concomitant formation of a tetranuclear cluster [Ni(3)(mu-ndt)(4)FeI] (2). Treatment of 1 with Na[N(SiMe(3))(2)] transforms some of the CO ligands into CN(-), and the monocyanide complex (PPh(4))[(CO)(2)(CN)Fe(mu-ndt)Ni(mu-ndt)Fe(CO)(3)] (3) and the dicyanide complex (PPh(4))(2)[(CO)(2)(CN)Fe(mu-ndt)Ni(mu-ndt)Fe(CO)(2)(CN)] (4) were isolated. X-ray structural analyses of the trinuclear complexes revealed a Fe-Ni-Fe array in which the metal centers are connected by the ndt sulfur bridges and direct Fe-Ni bonds. Hydrogen bonding between the CN ligand in 3 and cocrystallized ethanol was found in the solid-state structure. The monocyanide complex 3 and dicyanide complex 4 reacted with acids such as HOTf or HCl generating insoluble materials, whereas complex 1 did not react.

Abstract  The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.

Abstract  Two PRL-like glycoprotein hormone complexes were purified from the medium of cultured mouse conceptuses from day 10 of pregnancy: mouse placental lactogen-I (mPL-I) (29-32K), and mPL-I (36.5-42K). Sodium dodecyl sulfate-gel electrophoresis revealed that mPL-I (36.5-42K) is a complex of five proteins with mol wt of 36.5K, 37.5K, 39K, 40.5K, and 42K. Deglycosylation with peptide: N-glycosidase F or trifluoromethanesulfonic acid produced a single 29K protein. mPL-I (36.5-42K) was also sensitive to neuraminidase, but not to endo-beta-N-acetylglucosaminidase H or bacterial alkaline phosphatase. The production of intermediates from partial digestion of mPL-I (36.5-42K) with endo-beta-N-acetylglucosaminidase F indicated the presence of multiple glycosylation sites. mPL-I (29-32K) is a complex of three proteins with mol wt of 29K, 30.5K, and 32K. Treatment with peptide:N-glycosidase F or trifluoromethanesulfonic acid reduced the mol wt of the 30.5K and 32K bands to 28K. The 30.5K band was sensitive to endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, but the 32K band was not. Neither band was sensitive to neuraminidase or bacterial alkaline phosphatase. The 29K band was resistant to all chemical and enzymatic treatments and is probably not glycosylated or phosphorylated. In the nonreduced state, neither form of mPL-I showed an increase in mobility over that of its reduced counterpart on sodium dodecyl sulfate-gel electrophoresis, indicating that neither form of mPL-I contains the large disulfide loop common to hormones of the PRL family. After iodination, all component proteins of both forms of mPL-I were found to bind to day 17 pregnant mouse liver membranes and were displaceable by excess mPL-II. In a radioreceptor assay, 125I-labeled mPL-I (36.5-42K) was displaced by mPRL or mPL-II, but not by mGH. An antiserum to both forms of mPL-I was generated, and a RIA employing mPL-I (36.5-42K) as the standard and radioligand was developed. Dilutions of day 10 pregnant maternal mouse serum and placental homogenate and a partially purified fraction of mPL-I (29-32K) produced displacement curves parallel to that of mPL-I (36.5-42K) standard curve. Five micrograms of mPRL, mPL-II, or mGH or 10 microliter day 17 pregnant or male mouse serum did not displace the radioligand from the antibody. mPL-I (36.5-42K) was lactogenic, but it did not possess LH-like bioactivity.

Abstract  Although sulfolane proved unexpectedly to be a poor solvent for solution-phase secondary-ion mass spectrometry of underivatized amino acids in the presence of thallium(I) salts, glycerol was somewhat more effective. Also, the addition of trifluoromethanesulfonic acid proved more effective than addition of the metal in generating molecular ion complexes. A convenient and reliable method for rapidly determining amino acid molecular ions is based on these observations.

Abstract  The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.

Abstract  Bioside Gal beta 1-3GalNAc alpha 1-O(CH2)3NHCOCF3 has been synthesized. The key alpha-glycoside GalNAc alpha 1-O(CH2)3NHCOCF3 (peracetate) was obtained either by isomerization of its beta-anomer with trifluoromethanesulfonic acid, or by direct glycosylation of 3-(trifluoroacetamido)propanol with D-galactosamine (anomeric pentaacetate) in the presence of a mixture of trifluoromethanesulfonic acid and boron trifluoride etherate. De-O-acetylated alpha-galactosaminide obtained was further transformed into benzylidene derivative, the latter was glycosylated with acetobromogalactose to give the protected alpha-bioside. The removal of the protecting groups gave the (3-aminopropyl)-alpha-bioside, which was subsequently immobilized on bovine serum albumin and cytochrome c.

Abstract  The biosynthesis and processing of cellulase from ripening avocado fruit was studied. The mature protein is a glycoprotein, as judged by concanavalin A binding, with a molecular weight of 54,200. Upon complete deglycosylation by treatment with trifluoromethane sulfonic acid the mature protein has a molecular weight of 52,800 whereas the immunoprecipitated in vitro translation product has a molecular weight of 54,000. This result indicates that cellulase is synthesized as a large molecular weight precursor, which presumably possesses a short-lived signal peptide. A membrane-associated and heavily glycosylated form of the protein was also identified. This putative secretory precursor was enzymically active and the carbohydrate side chains were sensitive to endoglycosidase H cleavage. Results of partial endoglycosidase H digestion suggest that this precursor form of the mature glycoprotein possesses two high-mannose oligosaccharide side chains. The oligosaccharide chains of the mature protein were insensitive to endoglycosidase H cleavage, indicating that transport of the membrane-associated cellulase to the cell wall was accompanied by modification of the oligosaccharide side chains. The presence of a large pool of endoglycosidase H-sensitive membrane-associated cellulase (relative to an endoglycosidase H-insensitive form) suggest that transit of this protein through the Golgi is rapid relative to transit through the endoplasmic reticulum.

Abstract  The heptacosapeptide amide corresponding to the entire amino acid sequence of chicken gastrin-releasing peptide (cGRP) was synthesized similarly to the synthesis of porcine GRP by assembling six peptide fragments followed by deprotection with 1 M trifluoromethanesulfonic acid-thioanisole in TFA. A new carboxyl-activating reagent, thiazolidine-2-thione, was preferentially adopted for preparation of necessary fragments. The synthetic cGRP, purified by ion-exchange chromatography, followed by partition chromatography, was active as the synthetic porcine GRP, when plasma immunoreactive gastrin level was examined in rats. No obvious difference was observed when synthetic and natural cGRP preparations were compared by HPLC, immunochemical property and biological activity in dogs.

Abstract  A position isomer of ganglioside GD3 has been synthesized in which N-acetylneuraminic acid (Neu5Ac) is linked alpha-glycosidically at C-9 of the Neu5Ac residue of the ganglioside GM3, structure. The coupling of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-beta-D-galactopyranosyl)-(1----4)-2,6-di-O-benzoyl-beta-D -glucopyranoside with methyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2-nonulopyranosylonate)-(2----9)-(methyl 5-acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-2-thio-D-glycero-D-galacto-2 -nonulopyranosid)onate, prepared from the corresponding 2-(trimethylsilyl)ethyl glycoside by selective removal of the 2-(trimethylsilyl)ethyl group, 1-O-acetylation, and introduction of the methylthio group with trimethyl(methylthio)silane, using N-iodosuccinimide-trifluoromethanesulfonic acid as a glycosylation catalyst, gave a tetrasaccharide (5). Compound 5 was converted, via O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and subsequent imidate formation, into a protected alpha-Neu5Ac-(2----9)-alpha-Neu5Ac-(2----3')-alpha-lactosyl trichloroacetimidate (8). Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 8 afforded a ceramide precursor which was transformed, via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and hydrolysis of the methyl ester groups, into to the little ganglioside.

Abstract  Hydroamination of alkenes or alkynes is one of the most straightforward methods to form C-N bonds and nitrogen-containing heterocycles. A simple Lewis acid Al(OTf)3 was found to be an effective precatalyst for the hydroamination of unactivated primary and secondary alkenylamines between 110 and 150 °C. Subsequent studies show that other metal triflates are also effective precatalysts for the hydroamination reactions. For metal triflate salts, mechanistic studies, including kinetics, are consistent with the in situ generation of triflic acid, which likely serves as the active catalyst.

Abstract  Proteins associated with the cell wall peptidoglycan (CW-Pr) of Mycobacterium tuberculosis H37Ra were isolated to evaluate their immunoreactivity and immunoprophylactic properties against experimental tuberculosis. Chemical treatment of the cell wall with trifluoromethanesulfonic acid: anisole (2:1) resulted in the release of three proteins of 71, 60 and 45 kDa as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparative study of immune responses elicited to individual proteins in mice immunized with CW-Pr emulsified in incomplete Freund's adjuvant showed the 71-kDa protein to be the most immunoreactive antigen. This 71-kDa protein was found to cross-react with the 70-kDa heat shock protein from M. leprae and possessed ATPase activity. Mice immunized with the 71-kDa protein exhibited significantly higher immune responses, on the basis of T and B cell reactivity, as compared to a M. bovis Bacillus Calmette Guerin (BCG)-vaccinated group. The culture supernatants collected from 71-kDa stimulated lymphocytes stimulated exhibited increased interferon-gamma and interleukin-2 production. The protective efficacy of the 71-kDa protein in comparison to BCG was determined by challenging the mice with a virulent strain M. tuberculosis H37Rv. The 71-kDa protein was found to be more protective in animals challenged at 8 and 16 weeks post immunization, shown by increased survival rates and decreased viable bacilli counts in the target organs as compared to BCG-vaccinated animals.

Abstract  Sequential methylation of arachno-6,9-C2B8H14 (1) led to a series of methyl derivatives and finally to the camouflaging of all boron positions by mixed persubstitution. Thus, deprotonation of 1 produced the [arachno-6,9-C2B8H13] anion (1(-)), the methylation of which with MeI in tetrahydrofuran proceeded on the open-face boron vertexes with the formation of 5-Me-arachno-6,9-C2B8H13 (2; yield 28%) and 5,8-Me2-arachno-6,9-C2B8H12 (3; yield 36%). Observed in this reaction was also a side formation of 2-Me-closo-1,6-C2B8H9 (4; yield 6%).The electrophilic AlCl3-catalyzed CH3(+) attack of the neutral 1 in neat MeI at ambient temperature afforded 1,3-Me2-arachno-6,9-C2B8H12 (5), while a 76-h heating at 120 °C generated a mixture of the di- and triiodo derivatives 1,2,3,4,8,10-Me6-5,7-I2-arachno-6,9-C2B8H6 (6) and 1,2,3,4,7-Me5-5,7,10-I3-arachno-6,9-C2B8H6 (7). On the other hand, a HOTf-catalyzed reaction between 1 and MeOTf at reflux resulted in the isolation of 2-TfO-1,3.4,5,7,8,10-Me7-arachno-6,9-C2B8H6 (8; Tf = CF3SO2; yield 65%). The compounds were characterized by multinuclear ((11)B, (1)H, (13)C, and (19)F) NMR spectroscopy, mass spectrometry, and elemental analysis, and the structures of compounds 1, 1(-), 5, and 6 were established by X-ray diffraction analysis.

Abstract  Cell surface hydrophobicity influences adhesion and virulence of the opportunistic fungal pathogen Candida albicans. Previous studies have shown that cell surface hydrophobicity is due to specific proteins that are exposed on hydrophobic cells but are masked by long fibrils on hydrophilic cells. This observation suggests that hydrophobic cell wall proteins may contain little or no mannosylation. In the present study, the glycosylation levels of three hydrophobic cell wall proteins (molecular mass range between 36 and 40 kDa) derived from yeast cells were examined. One hydrophilic protein (90 kDa) was also tested. Various endoglycosidases (endoglycosidase F-N-glycosidase F, O-glycosidase, beta-mannosidase, N-glycosidase F), an exoglycosidase (alpha-mannosidase), and trifluoromethane sulfonic acid were used to deglycosylate the proteins. All four proteins were reactive to the lectin concanavalin A, demonstrating that they were mannoproteins. However, gel electrophoresis of the control and treated proteins revealed that mannosyl groups of hydrophobic proteins were less than 2 kDa in size, while the mannosyl group of the hydrophilic protein had a molecular mass of approximately 20 kDa. These results suggest that unlike many hydrophilic proteins, hydrophobic proteins may have low levels of glycosylation. Changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface.

Abstract  Anion exchange chromatography resolves two charge variants of rat kidney endopeptidase-24.11 (designated NEP 1 and NEP 2); each was purified to homogeneity using immunoaffinity chromatography. In addition to charge differences the subunit molecular weights of NEP 1 and NEP 2 differ and are 89 and 96 kDa, respectively. Isoelectric focusing resolved 8-10 pl species in the pH range of 5.95-6.20 for NEP 1 and 5.46-6.06 for NEP 2. Removal of sialic acid residues converted the multiple pl species to one form with a pl of 6.32 for NEP 2, and two forms with pls of 6.27 and 6.32 for NEP 1. Endoglycosidase H or F, capable of removing high-mannose and biantennary branched N-linked oligosaccharides, produced a 2-3 kDa decrease in the molecular weight of both NEP 1 and NEP 2. Peptide-N-glycosidase F, capable of removing all classes of N-linked oligosaccharides, produced 8 and 11 kDa decreases in NEP 1 and NEP 2, respectively. Removal of all N-linked and O-linked oligosaccharides with trifluoromethanesulfonic acid resulted in 10 and 15 kDa decreases in NEP 1 and NEP 2, respectively. Tryptic epitope maps demonstrated that NEP 2 was cleaved at a slower rate than NEP 1. These analyses demonstrate that rat kidney NEP exhibits sialic acid microheterogeneity resulting in two distinct change variants. The data also indicate that NEP 2 contains more N- and O-linked carbohydrate mass than NEP 1 and may contain a larger polypeptide backbone giving rise to molecular weight differences between these enzyme forms.

Abstract  The reactivity of nitronium tetrafluoroborate in the nitration of deactivated di- and trifluoronitrobenzenes is enhanced in superacidic trifluoromethanesulfonic (triflic) acid compared with aprotic methylene chloride and sulfolane solutions. The enhanced reactivity is discussed in terms of better solubility and higher dissociation of the nitronium salts, as well as protosolvation of NO2+ by superacids.

Abstract  β,γ-Unsaturated ketones are an important class of organic molecules. Herein, copper catalysis has been developed for the synthesis of β-γ-unsaturated ketones through 1,2-addition of α-carbonyl iodides to alkynes. The reactions exhibit wide substrate scope and high functional group tolerance. The reaction products are versatile synthetic intermediates to complex small molecules. The method was applied for the formal synthesis of (±)-trichostatin A, a histone deacetylase inhibitor.

Abstract  alpha-Amylases from germinated maize, oats, rice, and sorghum were isolated by glycogen precipitation and hydrophobic interaction chromatography. Several methods were used for the detection of glycoproteins, including barley alpha-amylase isozymes purified as previously described and using the rice alpha-amylase as a positive control for glycosylation. Affinoblotting using concanavalin A, immunoblotting using a xylose-specific serum which reacts with complex N-linked glycans, and endo-beta-N-acetylglucosaminidase H treatment of amylases gave negative results for maize, oats, sorghum, and barley. However, after deglycosylation with trifluoromethanesulfonic acid, the molecular weight of one maize alpha-amylase constituent was clearly decreased. The same result was obtained after beta-elimination in mild conditions. Together these results indicated probable O-linked glycosylation of one maize alpha-amylase when barley, oats, and sorghum alpha-amylases did not appear to be glycosylated. Chemical deglycosylation of rice alpha-amylase resulted in the production of two polypeptides with different molecular weights.

Abstract  1-(Diphenylphosphoryl)alka-1,2-dienes (phosphonoallenes) in Brønsted (super)acids (TfOH, FSO3H, and H2SO4) at -70 to 120 °C for 30 min to 4 h gave, at first, (3-hydroxyalk-1-en-1-yl)diphenylphosphine oxides, as kinetically favorable reaction products, that are further converted into 1-phenyl-1,4-dihydrophosphinoline 1-oxides as thermodynamically stable compounds. The latter compounds are formed from phosphonoallenes under the action of a strong Lewis acid AlCl3 at room temperature for 10-120 min. This is a novel, simple and efficient (short reaction time, high yields) method for synthesis of such 1,4-dihydrophosphinoline 1-oxides.

Abstract  A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.

Abstract  Affinity-purified human testosterone-binding globulin (hTeBG) is composed of two subunits [mol wt (Mr), 52,200 and 48,600], as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, and immunochemical localization with a monoclonal antibody raised against rat androgen-binding protein. Fluorography of SDS-PAGE gels on which photoaffinity-labeled hTeBG was analyzed yielded essentially identical results. Enzymatic deglycosylation of hTeBG with neuraminidase to remove sialic acid led to the production of two subunits of 50,800 and 47,300 Mr when assessed by SDS-PAGE. Treatment of hTeBG with an optimal concentration of N-glycanase to remove Asn-linked oligosaccharides produced a single subunit of 44,100 Mr. When hTeBG was treated with neuraminidase and O-glycanase to remove O-linked oligosaccharides, three subunits were seen, two of which had Mr not clearly different from those obtained with neuraminidase treatment alone plus a subunit of 40,900 Mr. Treatment of hTeBG with a combination of all three enzymes produced a single subunit of 42,900 Mr. Chemical deglycosylation with trifluoromethane-sulfonic acid produced a single subunit with a Mr identical to that produced by treatment with all three enzymes. We concluded that this is the Mr of completely deglycosylated hTeBG. Based on this Mr, carbohydrates contribute 18% and 12% to the apparent Mr of the heavy and light subunits of hTeBG, respectively. Two-dimensional PAGE analysis of hTeBG with its oligosaccharides intact indicated that the heavy subunit was composed of seven isoelectric variants with pI values of 5.87-6.55, while the light subunit was composed of four charge variants with pI values of 6.14-6.55. Treatment of hTeBG with the enzymes resulted in a shift in the pH values to a more basic pH range, indicating that carbohydrate removal also removed charged species from the protein. The greatest cathodal shift occurred when hTeBG was treated with a combination of the three enzymes (pI 7.33-7.77) or when it was chemically deglycosylated (pI 6.37-7.02). Despite the apparent removal of all carbohydrates, the single subunit was still composed of multiple isoforms. This finding suggests that other charged species remain on the hTeBG molecule.

Abstract  The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.

Abstract  We have previously identified a molecule on the T cell surface, which, in addition to CD2 is involved in the rosette phenomenon. This is a 32-kDa single polypeptide chain which we have termed E2. The studies reported here show striking patterns on the glycosylation status of E2. It is a heavily sialylated and glycosylated molecule, the sugar moieties accounting for almost half of its relative molecular mass (Mr). It carries no N-linked sugar residues, only O-linked oligosaccharides. Despite heavy glycosylation, the molecule appears to behave homogeneously on gel electrophoresis, both in terms of Mr and pI. Neuraminidase treatment of E2 lowered its Mr to 28 kDa; this was further decreased to 18 kDa after removal of O-linked sugar residues by treatment with O-glycanase. An identical reduction in size was observed after treatment with trifluoromethane sulfonic acid, showing that the molecule carries no detectable N-linked sugar residues. Moreover, endoglycosidase F and endoglycosidase H treatment of either the immunoprecipitates from 125I surface-labeled thymocytes, or of a purified preparation of E2, did not reduce the Mr of E2, nor did tunicamycin treatment of T cells. Two-dimensional gel electrophoresis revealed two discrete spots of acidic pI (4.4 and 4.6) that were still seen after neuraminidase treatment, though they had moderately shifted. Pulse-chase experiments revealed a single 28-kDa precursor form that could have been the unsialylated molecule. Finally, sequencing 14 amino acid residues of the N-terminal side revealed no homology with known proteins. Since the sugar moieties of adhesion protein could play an important role, the results obtained in this study will prove valuable to our understanding of the role exerted by the E2 molecule.

Abstract  Highly glycosylated regions of mucins, or glycopeptides, were obtained by proteolysis of human bronchial mucins. They were deglycosylated by treatment with a trifluoromethane sulfonic acid/anisole mixture and subsequent solvolysis with anhydrous liquid hydrogen fluoride. The resulting peptides were then used to raise an immune serum in rabbit. This immune serum was used to localize the peptide precursors of human respiratory mucins within bronchial cells, using an immunohistochemical method. Two main patterns of labeling were observed in the goblet cells: the entire cytoplasm of some goblet cells was immunoreactive, whereas in other cells the labeling was concentrated around the nucleus. In the respiratory mucous glands, the labeling was localized around or below the nucleus. The serous cells were not stained. Similar labeling was observed in human colon goblet cells. This immune serum seems to be specific for mucin-secreting cells and has a strong affinity for the perinuclear region of these cells.