Affinity chromatography of antibodies directed against soybean lipoxygenase-1 and -2 and an enzyme-linked immunosorbent assay (ELISA) for antibodies and lipoxygenases

Crude immunoglobulin G (IgG) fractions of antisera directed against soybean lipoxygenase-1 and -2 were purified by being passed through an immunoadsorbent column containing lipoxygenase coupled to CNBr-activated Sepharose 4B. Bound immunoglobulin was desorbed with pulses of 2 M or 3 M ammonium thiocyanate or 0.1 M glycine-HCl buffer (pH 2.5). The total column recoveries of anti-lipoxygenase-1 IgG and anti-lipoxygenase-2 IgG were 45% and 58%, respectively. The affinity for lipoxygenase of immunospecific antibodies was determined in an enzyme-linked immunosorbent assay (ELISA). In a reaction with lipoxygenase-1, anti-lipoxygenase-1 IgG, which was eluted with glycine-HCl buffer (pH 2.5) with recovery of 24%, had a 6.5-times higher affinity than the whole IgG fraction of antiserum. The affinity of anti-lipoxygenase-2 IgG for lipoxygenase-2 increased 2.2-times after chromatography of IgG over an immunoadsorbent column using 2 M ammonium thiocyanate as eluent (recovery 21%).     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Isolation and structural studies of phosphate-containing oligosaccharides from alkaline and acid hydrolysates of Streptococcus pneumoniae type 6B capsular polysaccharide

The capsular polysaccharide of Streptococcus pneumoniae serotype 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----4)- D-RibOH-(5-P----]n was depolymerised under alkaline (NaOH) and acidic (HF) conditions. The former treatment yielded, as the major component, alpha-2-P-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-5- P-RibOH. The latter treatment at -16 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH-(5-P----2)- alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH and at 4 degrees gave alpha-Galp-(1----3)-alpha-Glcp-(1----3)-alpha-Rhap-(1----4)-Rib OH. These oligosaccharides were characterised by sugar analysis, f.a.b.-m.s., and 1H- and 13C-n.m.r. spectroscopy.     

Chemical behaviour of cytidine 5'-monophospho-N-acetyl-beta-D-neuraminic acid under neutral and alkaline conditions

The chemical behaviour of CMP-N-acetylneuraminic acid under neutral and different alkaline conditions has been investigated. The products formed were isolated by ion-exchange chromatography and gel filtration and analysed by colorimetric methods, thin-layer chromatography, combined gas-liquid chromatography/mass spectrometry and/or 360-MHz 1H-NMR spectroscopy. A maximum stability of CMP-N-acetylneuraminic acid was observed at pH8-11. In the tested pH range of 6-13, CMP and N-acetylneuraminic acid were formed in variable amounts as decomposition products. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid was produced at pH greater than 7; the amount of this substance increased with increasing pH. In anhydrous triethylamine its yield was 50%. A new neuraminic acid derivative, N-acetyl-beta-D-neuraminic acid 2-phosphate, could be isolated from the mixture of alkaline decomposition products of CMP-N-acetylneuraminic acid. The yield of this compound was maximum 22% in anhydrous triethylamine. Because 2-deoxy-2,3-dehydro-N-acetylneuraminic acid was formed under simulated physiological conditions, it is assumed that this compound, which occurs in tissues and fluids of man and animals, is derived from CMP-N-acetylneuraminic acid non-enzymically also under conditions in vivo.     

The structure of sialyl-glycopeptides of the O-glycosidic type, isolated from sialidosis (mucolipidosis I) urine

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+-translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean-acid-metabolism plant Kalancho? daigremontiana and compare it with other phosphohydrolases. Vacuoles were isolated by polybase-induced lysis of mesophyll-cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m-3), but was not completely inhibited even at much higher ammonium-molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m-3 ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m-3. This is taken as evidence that the vacuolar ATPase might function as an H+-translocating ATPase. It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic-acid accumulation in K. daigremontiana.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Primary structure of two sialylated triantennary glycans from human serotransferrin

Glycopeptides obtained from human serotransferrin by pronase digestion were separated into two fractions by affinity chromatography on Con A-Sepharose. The retarded fraction (85% of total glycopeptides) contained sialylated biantennary glycans of the N-acetyllactosaminic type, the primary structure of which has been previously determined. The non-retained fraction (15% of total glycopeptides) consisted of two isomeric triantennary glycans of the N-acetyllactosaminic type. The primary structure have been elucidated by methylation analysis and 500 MHz 1H-NMR spectroscopy. Both contain an additional NeuAc(alpha 2----3)Gal(beta 1----4)GlcNAc antenna. The latter is linked to C-4 of the (alpha 1----3) bound Man residue in 45% of the glycans in the non-retained fraction but to C-6 of the (alpha 1----6) bound Man residue, in the remaining 55% of the glycans in this fraction.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.