Reverse phase HPLC fractionation of the oligosaccharide alditols isolated from an I-active ovarian cyst mucin glycoprotein

We have used reverse phase high pressure liquid chromatography (HPLC) to separate the reduced oligosaccharides produced by alkaline borohydride degradation of ovarian cyst blood group substances. From a single cyst, six oligosaccharides, ranging from two to seven residues in length, have been isolated by preparative HPLC on C-18 stationary phases using water for elution. The purity of the products and their structures were determined by high field proton NMR spectroscopy in conjunction with exo-and endoglycosidase digestion. All the chains isolated terminated inN-acetylgalactosaminitol which was substituted at the 3-postion by galactose and in some cases at the 6-postion byN-acetylglucosamine. The largest identified oligosaccharide was a heptasaccharide alditol containing a single -linked fucose in a Lewis blood group structure (Le^a).     

Immunochemical studies on blood groups. Immunochemical properties of B-active and non-B-active blood group substances from horse gastric mucosae and the relative size distributions of oligosaccharides liberated by base-borohydride.

Horse B-active and non-B-active glycoproteins from gastric mucosae are indistinguishable in their precipitating abilities with concanavalin A, anti-BP1, type XIV horse antipneumococcal serum, the lectin from Lotus tetragonolobus and a group 1 anti-I serum, Ma; no Le^a or Le^b activity was found. Each was subjected to catalyzed release of its oligosaccharide chains by 0.05 n NaOH in 1 m NaBH₄. Destruction of serine, threonine and 2-acetamido-2-deoxygalactopyranose (dGalNAc) was associated with production of alanine, α-aminobutyric acid and N-acetyl-d-galactosaminitol, as expected for a carbohydrate to peptide linkage via dGalNAc to serine or threonine. No evidence of basecatalyzed peeling was seen. Bio-Gel P-2 elution patterns of the salt-free oligosaccharides from the two preparations were compared. Unlike results obtained with human ovarian cyst substances, very little material was excluded. The largest-size chains are in the range of deca- or dodecasaccharides, and a reduced octasaccharide was isolated. The four most abundant amino acids in both B-active and non-B-active materials are threonine, serine, proline and glutamic acid, which together account for 60% of the weight of amino acids.     

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues

We describe here the synthesis of the allyl Le^a trisaccharide antigen as well as that of an analogue of the Le^x trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le^a analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the α-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF₃·OEt₂ than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF₃·OEt₂, glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

The lectin from Euonymus europeus seeds was purified by adsorption onto insoluble polyleucyl hog A + H blood group substance and subsequent elution with lactose. The isolated lectin formed three lines in immunoelectrophoresis against rabbit antisera to the crude seed extract and showed three components on electrophoresis in acrylamide gel at pH 9.4. In analytical isoelectric focusing the purified lectin had six closely spaced bands with pI from 4.3 to 4.7. It sedimented as two peaks: a big symmetrical peak with s_20,w⁰ of 7.8 and another small, diffuse moving peak. The intrinsic viscosity was 0.057 dl/g and the M_r calculated from the sedimentation coefficients, intrinsic viscosity, and V? of 0.71 was about 166,000. In sodium dodecyl sulfate, it gives subunits of M_r 17,000 and 35,000; 20% of the 35,000 subunit resists reduction by dithiothreitol in 7 m guanidine-HCl. The Euonymus lectin is a glycoprotein containing 4.8% d-galactose, 2.9% d-glucose, and 2.8% N-acetyl-d-glucosamine. The purified lectin precipitated well with B and H blood group substances and with the P1 fraction of blood group B substance but not with A₁ substances. It precipitated poorly with Le^a and Le^b and precursor I blood group substances. Inhibition of precipitation with milk and blood group oligosaccharides showed the lectin to be most specific for blood group B oligosaccharides having the structure: dGalα1 → 3[lFucα1 → 2]dGalβ1 → 3 or 4dGlcNAcβ→. It is also inhibited by blood group H oligosaccharides but to a lesser degree. For 50% inhibition of precipitation, 3.5, 850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively are required. The B and H specificities are an intrinsic property of a single lectin site since absorption and elution from an H immunoadsorbent gave material with B as well as H specificity. Millipore-filtered crude extracts of Euonymus europeus preserved with 0.02% sodium azide are stable in the refrigerator for many months and can be used for quantitative precipitin and for quantitative inhibition assays, results being the same as with purified lectin.     

Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.     

Immunochemical studies on blood groups. Structures and immunochemical properties of nine oligosaccharides from B-active and non-B-active blood group substances of horse gastric mucosae.

Oligosaccharides from base-borohydride-treated B-active and non-B-active glycoproteins of horse stomach mucosae were purified chromatographically on Bio-Gel P-2, charcoal-Celite, paper and high pressure liquid chromatography. From colorimetric and gas-liquid Chromatographic analyses, methylation, quantitative periodate oxidation and Smith degradation, structures of nine Oligosaccharides are proposed. Seven have not been previously described. The oligosaccharide isolated in largest amount in the B-active reduced tetrasaccharide analogous to an A-active reduced oligosaccharide from pig submaxillary mucin, and a reduced octasaccharide, the largest isolated, has two B determinants and may represent full expression of B-specific biosynthetic potential of the mucosal lining. Three B-active and one non-B-active oligosaccharide possessed the core structure previously identified in Oligosaccharides from human blood group, B, HLe^b, Le^a and precursor I substances. Two non-B-active and one B-active compound inhibited the cross reaction of type XIV horse antipneumococcal sera with blood group substances. Terminal nonreducing α-linked dGlcNAc (d-2-acetamido-2-deoxyglucopyranose), previously found in Oligosaccharides of hog blood group substances, was also present in a tetrassarcharide of the non-B-active material. Oligosaccharides released from blood group glycoproteins of horse stomach mucosae are smaller and hence less heterogeneous than those from human ovarian cyst and perhaps hog A + H and human gastric mucosae.     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [α-L-Fucp-(1-2)-β-D-Galp-(1-4)-D-Glc, K _a = 4.2 × 10³ M^?1]. The following antigens were bound with a weaker affinity: H-disaccharide α-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Le^y α-L-Fucp-(1-2)-β-D-Galp-(1-4)-[α-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (K _a 1.1?1.7 × 10³ M^?1). The lowest binding was observed for L-fucose (K _a = 2.7 × 10² M^?1) and trisaccharide Le^a, (β-Galp-(1-3)-[α-L-Fucp-(1-4)]-GlcNAc (K _a = 6.4 × 10² M^?1). The Le^d, Le^a, and Le^x pentasaccharides and Le^b hexasaccharide were not bound to LABA.     

Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

Immunochemical studies on blood groups: The internal structure and immunological properties of water-soluble human blood group A substance studied by Smith degradation,liberation, and fractionation of oligosaccharides and reaction with lectins

Carbohydrate structures in the interior of a blood group A active substance (MSS) were exposed by one and by two Smith degradations. Reactivities of the original glycoprotein and its Smith degraded products with 13 different lectins and with anti-I Ma were studied by quantitative precipitin assay. MSS and its first Smith degraded product completely precipitated Ricinus communis hemagglutinin with five times less of the first Smith degraded glycoprotein being required for 50% precipitation. The second Smith degraded material precipitated only 90% of the lectin. MSS did not precipitate peanut lectin, whereas its first and second Smith degraded products completely precipitated the lectin. The first Smith degraded glycoprotein also reacted well with Wistaria floribunda, Maclura pomifera, Bauhinia purpurea alba, and Geodia lectins indicating that its carbohydrate moiety could contain dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc, dGalβ1 → 3dGlcNAcβ1 → 3dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc determinants at nonreducing ends. The second Smith degraded material precipitated well with Ricinus communis hemagglutinin, Arachis hypogaea, Geodia cydonium, Maclura pomifera, and Helix pomatia lectins showing that dGalNAc, dGalβ1 → 3dGalNAc, dGalβ1 → 4dGlcNAc residues at terminal nonreducing ends could be involved. Monoclonal anti-I Ma (group 1) serum reacted strongly with the first Smith degraded product indicating large numbers of anti-I Ma determinants, dGalβ1 → 4dGlcNAcβ1 → d 6dGal and/or dGalβ1 → 4dGlcNAcβ1 → 6dGalNAc at nonreducing ends. The comparable activities of the native and Smith degraded products with wheat germ lectin indicate capacity to react with DGlcNAc residues at nonreducing ends and/or at positions in the interior of the chain. The totality of lectin reactivities indicates heterogeneity of the carbohydrate side chains. Oligosaccharides with ³H at their reducing ends released from the protein core of the first and second Smith degraded products were obtained by treatment with 0.05 m NaOH and 1 M NaB³H₄ at 50 °C for 16 h (Carlson degradation). The liberated reduced oligosaccharides were fractionated by dialysis, followed by retardion, Bio-Gel P-2, P-4, and P-6 columns. They were further purified on charcoal-celite columns, and by preparative paper chromatography and high-pressure liquid chromatography. Their distribution by size was estimated by the yields on dialysis, Bio-Gel P-2, and Bio-Gel P-6 chromatography, and from the radioactivity of the reduced sugars. Of the oligosaccharide fractions from the first Smith degraded product, about 77% of the carbohydrate side chain residues contained from 1 to 6 sugars, 13% from 7 to perhaps 12 sugars, and 10% was nondialyzable (polysaccharides and glycopeptide fragments). Of the second Smith degraded product, approximately 82% of carbohydrate residues had from 1 to 6 sugars, 14% from 7 to perhaps 20 sugars and 4% was nondialyzable. The biological activity profile of the two Smith degraded products together with the size distributions of the oligosaccharides indicated that their carbohydrate side chains, comprised a heterogeneous population ranging in size from 1 to about 12 sugars. When most of these chains that are shorter than hexasaccharides are fully characterized it may be possible to reconstruct the overall structure of the carbohydrate moiety of the blood group substances and account for their biological activities.     

Synthesis of a BSA-Lex glycoconjugate and recognition of Lex analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross reactive analogue

A Le^x trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF₃·OEt₂. In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate–mediated coupling. This BSA-Le^x glycoconjugate displayed 35 Le^x haptens per BSA molecule. The relative affinity of the anti-Le^x monoclonal antibody SH1 for the Le^x antigen and analogues of Le^x in which the d-glucosamine, l-fucose or d-galactose residues were replaced with d-glucose, l-rhamnose and d-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Le^x antigen, only the analogue of Le^x in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Le^x analogues by the anti-Le^x mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le^x antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Le^x antigen. The identification of a trisaccharide analogue that does not cross-react with Le^x but still retains the same conformation as Le^x constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le^x tumor associated carbohydrate antigen.     

Immunochemical studies on the reactivities and combining sites of the d-galactopyranose- and 2-acetamido-2-deoxy-d-galactopyranose-specific lectin purified from Sophora japonica seeds

Sophora japonica lectin agglutinates human B erythrocytes strongly and A₁ erythrocytes weakly. Bivalent metal ions such as Ca²⁺, Mn²⁺, or Mg²⁺ were shown to be essential for hemagglutinating and precipitating activities. At optimal concentrations of bivalent metal ions, hemagglutinating activity was highest between pH 8.5 and 9.0 and decreased sharply below pH 8.5, whereas precipitating capacity was maximal between pH 6.7 and 9.5. The combining site of the S. japonica lectin was explored by quantitative precipitin and precipitin inhibition assays. This lectin showed substantial differences in precipitation with several blood group B substances ascribable to heterogeneity resulting from incomplete biosynthesis of their carbohydrate side chains. The lectin precipitated moderately well with A₁ substance and precursor blood group I fractions (OG). It precipitated weakly or not at all with A₂, H, or Le^a substances. In inhibition assays, glycosides of dGalNAc were about five to six times better than those of dGal; dGalNAc itself was about six times better than dGal. Nitrophenyl glycosides were all substantially better than the methyl glycosides, indicating a hydrophobic contribution to the site subterminal to the nonreducing moiety. Although nitrophenyl β-glycosides were much better than the corresponding α-glycosides, the methyl α-and βDGalNAcp were equal in activity as were methyl α- and βDGalp. Among the oligosaccharides tested, the β-linked N-tosyl-l-serine glycoside of dGalβ1 → 3dGalNAc was best and was as active as p-nitrophenyl βDGalNAcp, whereas dGalβ1 → 3dGalNAc α-N-tosyl serine and the nitrophenyl and phenyl α-glycosides of dGalβ1 → 3dGalNAc were much less active, suggesting that the hydrophobic moiety and/or a subterminal dGalNAc β-linked and substituted on carbon 3 play an important role in binding and that a β-linked glycoside of dGalβ1 → 3dGalNAc may be an essential requirement for binding. The results of inhibition studies with other oligosaccharides indicate that a subterminal dGlcNAc substituted on carbon 3 or 4 by dGalβ may contribute somewhat to binding and that whether the dGlcNAc is linked β1 → 3 or β1 → 6 to a third sugar does not contribute to or interfere with binding. The β1 → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4dGlcNAc was one-half as active as the corresponding β1 → 3-linked compound and the subterminal sugar must be unsubstituted for optimal binding. N-Acetyllactosamine was 50% more active than lactose, indicating that the subterminal N-acetamido group was also contributing significantly to binding. A variety of other sugars, glycosides, and oligosaccharides showed little or not activity. From the oligosaccharides available, the combining size of this lectin would appear to be least as large a β-linked disaccharide and most complementary to dGalβ1 → 3dGalNAc β-linked to tosyl-l-serine the most active compound tested.     

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA₁ or its immediate precursors. The primary site of synthesis of GA₂₀ from GA₁₉ was immature leaflets and tendrils, and the synthesis of bioactive GA₁ and its inactive catabolite GA₈ occurred predominantly in stem tissue. GA₂₉, the inactive catabolite of GA₂₀, was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA₁, GA₁₉, or even GA₂₀. During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [³H]GA₁ (and [³H]GA₂₉) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA₂₀ 3β-hydroxylase protein.     

Purification and characterization of lectin II from Ulex europaeus seeds and an immunochemical study of its combining site.

The lectin II from Ulex europaeus seeds was purified by adsorption on insoluble polyleucyl hog A + H blood group substance and elution with 35% ethylene glycol, and by chromatography on ?-aminocaproyl-fucosyl-amine-agarose. In immunodiffusion against rabbit antiserum to the crude extract, the isolated lectin formed one line which fused with one of the five formed by crude extract. The purified lectin showed two bands on acrylamide electrophoresis under alkaline or acid conditions but only one band of molecular weight 23,000 if the electrophoresis was in the presence of 0.1% sodium dodecyl sulfate at pH 8.8. The agglutinating and precipitating abilities are abolished by EDTA and can be restored by bivalent cations. The purified lectin precipitated to different extents with blood group A₁, A₂, B, HLe^b, Le^a, and I precursor substances and with acid- or Smith-degraded substances. Inhibition of precipitation indicated that the lectin site was unusual in that it interacted most strongly with the h-specific oligosaccharide

and with 2′-fucosyllactose, followed by β1 → 4 linked oligomers of dGlcNAc. Molecular models showed that all these inhibitors have a similarity in three-dimensional structures that could account for their activities.     

A monoclonal antibody that recognizes both leb and Y (Ley) antigens

A hemagglutinating monoclonal antibody has been obtained from a mouse/mouse hybridoma after immunisation with the le^b-active oligosaccharide, lacto-N-difucohexaose I, coupled to edestin. The antibody agglutinated human red cells regardless of Lewis phenotype. Blood group O cells were strongly, agglutinated, and progressively weaker agglutination was observed with A₂, B and A₂B cells. Blood group A₁ and A₁B cells were not agglutinated.By examining the binding of the antibody to glycolipids and oligosaccharides it was shown that the Le^b and Y (Le^y)-haptens bind to a similar extent. Full binding activity was dependent on the presence of, both fucosyl residues.Abbreviations LND l lacto-N-difucohexaose l - IV²Fuc,lll⁴FucLcOse₄ LND l-OL, lacto-N-difucohexaitol l     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

Immunochemistry of the Lewis-blood-group system: Partial characterization of Lea-, Leb-, and H-type 1 (LedH)-blood-group active glycosphingolipids from human plasma

Four different H-type 1 (Le^dH) blood-group-active glycosphingolipids (Le^dH-I–IV) have been isolated from the plasma of blood-group O Le(a?b?) secretors. The agglutination of O Le(a?b?) erythrocytes from secretors by 50 μl of 4 hemagglutinating units of caprine anti-Le^dH (anti-H-type 1) serum was inhibited by 0.02 μg of each of all four glycolipids. No Le^a or Le^b activities or reaction against Ulex europaeus lectin could be found. Le^dH-I, -II, -III, and -IV at 0.05, 0.01, 0.01, and 0.02 μg each are sufficient for incubation in order to convert 9 × 10⁷ O Le(a?b?) erythrocytes from nonsecretors into H-type 1 (Le^dH)-positive cells. Structural analysis of the H-type 1 glycolipids was performed in comparison to that of Le^a- and Le^b-blood-group-active glycolipids from human plasma isolated previously: Gas chromatography of peracetylated alditols revealed sugar composition. Combined gas chromatography-mass spectrometry established the glycosidic linkages. Together with the results obtained by direct inlet mass spectrometry of permethylated glycosphingolipids and by 360-MHz ¹H nuclear magnetic resonance spectroscopy (Egge, H., and Hanfland, P., 1981, Arch. Biochem. Biophys., 210, 396–404; Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U., 1981, Arch. Biochem. Biophys., 210, 405–411) the complete structures of the oligosaccharide chains of the Le^a-, Le^b-, and H-type 1-active glycolipids were established: Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^a antigens; Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^b antigens; and Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the H-type 1 (Le^dH) glycolipids. The diverse antigens of the same blood-group specificity obviously differ from one another in their lipid residue. In addition, plasmatic neolactotetraosylceramide could be identified, differing from that of human erythrocytes by a slower migration behavior in thin-layer chromatography.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Metabolism of Tritiated Gibberellin A(9) by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₉ (³H-GA₉) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA₂₀, GA₁₀, 2,3-dihydro-GA₃₁, and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA₃₀ and 2,3-dihydro-GA₃₁ were produced within 5 hours following ³H-GA₉ application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after ³H-GA₉ application. Levels of GA₁₀ increased with time, and since no GA₁₀ was produced during the purification procedures, GA₁₀ was, in all probability, produced from ³H-GA₉ within the plant tissue. The radioactive interconversion products produced by pea from ³H-GA₉ have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of ³H-GA₉ with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).