Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

Summary The adamantane moiety was introduced in the tachykinin NK₂ receptor-selective agonist [-Ala⁸]-NKA(4–10) (H-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂, MEN 10210) and in different positions of the NK₂ receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂) in order to investigate how this substitution affects their biological activity at tachykinin NK₁, NK₂ and NK₃ receptors. 1-Adamantaneacetic acid (1-Ada-CH₂COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH₂CO-Asp-Ser-Phe-Val--Ala-Leu-Met-NH₂). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH₂CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH₂CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH₂CO)-NH₂]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH₂] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate -COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK₁ and NK₂ receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK₃ receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK₁, NK₂ and NK₃ receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK₂ receptors of the rabbit isolated pulmonay artery.     

Biological activity of some conjugated gibberellins

Summary The biological activity of several gibberellin (GA) conjugates was studied and compared with that of the corresponding free GAs. The following conjugates were included: O(3)--d-glucopyranosides of GA₁, GA₃ and GA₄; O(13)--d-glucopyranosides of GA₁, GA₃ and GA₅; O(13)--d-glucopyranosyl-GA₅--d-glucopyranosyl ester; GA₃--d-glucopyranosyl ester and GA₃--d-glucopyranosyl ester; N-GA₃-oyl-glycine, its methyl ester, N-GA₃-oyl-glycylglycine, and N-GA₃-oyl-proline. All compounds were synthesized chemically but some of them are known to occur as endogenous plant products, or to be formed in plants upon application of a free GA. Activity was determined in the dwarf pea, dwarf corn, dwarf rice, and lettuce hypocotyl bioassays. The GA conjugates were found to posses different relative activities depending on the chemical structure, the bioassay system, and the site of application (shoot or roots). It is concluded that the activity of GA conjugates as measured in different bioassays is based upon the ability of plant enzymes and possibly of certain microorganisms to hydrolyze glucosidic, glucosyl ester, and amide-like linkages.Gibberellins-XLVIII. Part XLVII=Adam et al. (1976b)     

Comparative immunological properties of enantiomeric peptides

Summary The immunogenicity of a peptide composed of only d-amino acids is compared with that of the corresponding l-peptide enantiomer. Following three administrations of 100 g of individual peptide formulated with different adjuvants (Freund's complete adjuvant, QS21, or alum) to BALB/c mice, guinea pigs and rabbits, the l-peptide elicited strong l-peptide-specific IgG antibody responses in all formulations, whereas the d-peptide-induced d-peptide-specific IgG antibodies in the Freund's complete adjuvant and QS21 formulations, but was nonimmunogenic in the alum formulation. Mouse T-cell lines induced by the d-peptide formulated in Freund's complete adjuvant were found to express significant amounts of IL-2 when they were stimulated by the d-peptide. When an equal amount of both enantiomers was mixed and administered in Freund's complete adjuvant, only an l-peptide-specific IgG antibody response was observed. These results suggest that (i) d-peptide is immunogenic when strong adjuvant is provided; (ii) the immune system has preferential recognition of l-amino acid peptide; and (iii) the d-peptide can elicit d-peptide-specific T-cell responses.     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

Solid-phase synthesis of human salivary mucin-derived O-linked glycopeptides

Summary Short glycopeptides derived from salivary mucin have been synthesized in order to delineate the O-glycosylation pattern that is important in the biological activity of mucin. Two glycopoptides, APPETT*AAP-OMe and PAPPSS*SAP-OMe (*=-d-GalNAc), were prepared by solid-phase peptide synthesis integrating the Fmoc and Boc strategies. Since these peptides contain a C-terminal proline, we devised an efficient strategy using facile Boc chemistry, where the glycosylation at the desired position in the sequence was achieved using corresponding Fmoc-glycoamino acid esters A and B as building blocks. The transformation of the 2-azido group into the acetamido derivative was performed with thioacetic acid on the polymer-bound glycopeptides. Corresponding nonglycosylated peptides were also synthesized to study the influence of -d-GalNAc on peptide backbone conformation.     

Sequence analysis ofp-hydroxyphenyl-O-β-d-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [³H]galactose, and free [³H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After¹²⁵I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J 269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG glycosaminoglycans - CS chondroitin sulfate - DS dermatan sulfate - Ser serine - Xyl d-xylose - Gal d-galactose - GlcA d-glucuronic acid - IdoA l-iduronic acid - GalNAc N-acetyl-d-galactosamine - GlcNAc N-acetyl-d-glucosamine - HexA 4-deoxy-l-threo-hex-4-enopyranosyluronic acid - HO-Phe p-hydroxyphenyl group - HO-Phe-Xyl p-hydroxyphenyl-O--d-xylopyranoside - O₂N-Phe-Xyl p-nitrophenyl--d-xylopyranoside - OSO₃ ester sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - FPLC fast performance liquid chromatography - LC standard liquid chromatography     

d-Xylanase produced by Schizophyllum radiatum

Summary d-Xylanase (1,4--xylan xylanohydrolase, EC 3.2.1.8) was obtained from mycelial submerged culture of the mushroom Schizophyllum radiatum, grown on wheat straw pretreated with steam explosion as the substrate. The enzyme was purified 192-fold (specific activity 455 IU mg^-1 protein), with 37% yield with respect to total d-xylanase activity. Polyacrylamide electrophoresis of the d-xylanase peak showed a single band of protein whose molecular weight, calculated by electrophoretic mobility, was 25 700. The enzyme exhibited maximum activity at pH 4.9 and 55°C. d-Xylanase was stable from pH 5.0 to 7.5; its half-life was 12 h at 45°C. The Michaelis constant was 9.5 mg ml^-1 and V _max 0.37 mole min^-1. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylobiose, d-xylotriose, d-xylotetraose, and d-xylopentose showing the mode of action of an endo-type enzyme.     

Studies on the nature of the Li locus in Trifolium repens L. II. The effect of genotype on enzyme activity and properties

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl -d-glucoside, linamarin-lotaustralin, and p-nitrophenyl -d-galactoside. These three substrates were used in tests on the effect of genotype on enzyme activity. An absence of dominance at the Li locus was demonstrated, with a dosage effect of Li alleles on enzyme activity. A new Li allele was identified in the Lili clone, C11, which had low levels of enzyme activity. In crosses with two lili clones, Li(C11)li progeny were produced with activity levels similar to those of the C11 parent. Inhibition and heat-inactivation tests suggest that the lili clone, D4, produces an altered form of -glucosidase which may also be present in LiLi plants. The nature of the Li locus is discussed.     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Structural analysis ofBacillus licheniformis 86 surfactant

Summary A tentative structure and composition of a surfactant, BL-86, produced byBacillus licheniformis 86 is described. The surfactant is a mixture of lipopeptides with the major components ranging in size from 979 to 1091 Da and varying in increments of 14 Da. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the surfactant. There are 7 amino acids per molecule. The peptide portion is composed of the following amino acids: glutamic acid or glutamine (glx), aspartic acid or asparagine (asx), valine, leucine, and isoleucine at a ratio of 1.01.01.43.00.6, respectively. The leucine is present as both thed andl isomers at a ratio of about 21, respectively. Forty percent of the molecules containl-valine instead ofl-isoleucine. The glx and asx are present as a combination ofl-glutamic acid andl-asparagine and/orl-glutamine andl-aspartic acid. The N-terminus of the peptide is blocked, most likely by a peptide bond to the lipid portion. An ester carbonyl structure is present, which could be a part of a lactone ring connecting the position of the lipid to one of the carboxyl groups in the peptide. The lipid portion is composed of, on average, 8–9 methylene groups, and contains a mixture of linear and branched tails. Results of DCI-MS and FAB-MS analyses, as well as surface tension measurements, of purified BL-86 HPLC fractions support the proposed composition.     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH₂ and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.     

Differential binding characteristics and applications of dGalβ1→3dGalNAc specific lectins

Summary The binding properties of Arachis hypogaea (PNA), Bauhinia vurpurea alba (BPL), Maclura pomifera (MPL) and Sophora japonica (SJL) lectins were studied by quantitative precipitin and precipitin inhibition assays, demonstrating them to be most specific for dGal13dGalNAc residues. Additionally, each lectin had its own binding characteristic such as different binding abilities to dGal14dGlcNAc or dGal13dGlcNAc1linked oligosaccharides, and/or dGalNAc1linked to the Ser or Thr of the protein moiety. These differential binding characteristics can be used for investigating fine differences of the carbohydrate structure of the glycoconjugates, especially those having dGal13dGalNAc residues as terminal non-reducing ends.Abbreviations dGal d-galactopyranose - dMan d-mannopyranose - dGalNAc 2-acetamido-2-deoxy-d-galacto-pyranose - dGlcNAc 2-acetamino-2-deoxy-d-glucopyranose - LFuc L-fucose - NeuNAc N-acetylneuraminic acid - Ser serine - Thr Threonine - RCA Ricinus communis agglutinin - SBA Soy bean agglutinin (Glycine max) - HPA Helix pomatia agglutinin - DBL Dolichos biflorus lectin - GCL Geodia cydonium lectin     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

The presence of an endogenous lectin in early embryos ofXenopus laevis

Summary Xenopus laevis embryos were examined for the presence of endogenous carbohydrate binding proteins. Soluble extracts of cleavage, gastrula and neurula embryos are able to agglutinate trypsinized rabbit erythrocytes. Unlike other embryonic lectins this agglutination activity requires the presence of calcium ions but not of sulphydryl reducing agents. It is specifically inhibited by galactose and galactose containing derivatives. Thiodigalactoside is the most potent disaccharide inhibitor followed by lactose and melibiose respectively. Methyl -d-galactopyranoside is a more effective inhibitor than its anomer. N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, methyl -d-mannopyranoside andl-fucose do not inhibit activity at concentrations at or above 25 mM. EDTA and EGTA are also strong inhibitors of this activity. -d-galactoside binding lectins present in the early chick embryo have been implicated in cell to cell and cell to substrate adhesiveness of extraembryonic chick endoderm cells. Since cells of the blastula inXenopus laevis possess surface receptors bearing terminal -d-galactoside groups it is possible that this -d-galactoside binding lectin may play a role in the control of cell surface mediated events during development.     

Formation of d-3-hydroxybutyryl-coenzyme A by an acetoacetyl-coenzyme A reductase in Syntrophomonas wolfei subsp. wolfei

Cell-free extracts of Syntrophomonas wolfei subsp. wolfei synthesized d-(-)-3-hydroxybutyryl-coenzyme A (CoA) (the stereoisomer required for the synthesis of poly--hydroxyalkanoate) from acetoacetyl-CoA, but not crotonyl-CoA, and NAD(P)H. Ammonium sulfate fractionation and ion exchange chromatography separated an acetoacetyl-CoA reductase activity that formed d-(-)-3-hydroxybutyryl-CoA from the -oxidation enzyme activity, l-(+)-3-hydroxyacyl-CoA dehydrogenase. The former activity was further purified by hydroxylapatite and affinity chromatography. The most pure acetoacetyl-CoA reductase preparations formed d-(-)-3-hydroxybutyryl-CoA from acetoacetyl-CoA and had high specific activities using either NADH or NADPH as the electron donor. Thus, S. wolfei makes d-(-)-3-hydroxybutyryl-CoA by an acetoacetyl-CoA reductase rather than by a d-isomer specific enoyl-CoA hydratase and the reducing equivalents required for PHA synthesis from acetoacetyl-CoA can be supplied from the NADH made during -oxidation.     

l-Sorbose metabolism in Agrobacterium tumefaciens

The pathway of l-sorbose metabolism in Agrobacterium tumefaciens strain B6 was determined to be: l-sorbose d-glucitol (sorbitol) d-fructose d-fructose-6-phosphate d-glucose-6-phosphate. The reduction of l-sorbose and the oxidation of d-glucitol were mediated by NADPH- and NAD⁺-linked oxidoreductases, respectively. The intermediates, d-glucitol and d-fructose, were isolated from in vitro reaction mixtures by column chromatography on Dowex 1-borate, and identified enzymatically. d-Fructose was identified chemically by its ¹H-NMR spectrum and the IR spectrum and the melting point of the fructosazone. d-Glucitol was characterized chemically by the melting point and the IR spectrum of its hexaacetate. A. tumefaciens ICPB TT111, a representative of another genetic race of Agrobacterium, lacked l-sorbose reductase and therefore failed to grow on l-sorbose; it grew normally on d-glucitol.     

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferase using synthetic glycopeptide substrates

The factors determining glycosylation of mucin type glycoproteins are not well understood. In the present work, we investigated the role of the peptide moiety and of the presence of O-glycan chains on O-glycosylation by UDP-GalNAc: polypeptide -N-acetylgalactosaminyl-transferase (ppGalNAc-T). We used purified ppGalNAc-T from bovine colostrum and a series of synthetic glycopeptide and peptide substrates most of which contained sequences derived from the tandem repeat region of MUC2 mucin. The rate of incorporation of GalNAc into Thr was significantly greater than toward Ser residues. The presence of one or two GalNAc-Thr moieties in the substrate significantly reduced enzyme activity, and this effect was more pronounced when the disaccharide Gal1–3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal1–3GalNAc-Thr occurs at a slower rate than primary glycosylation of carbohydrate-free peptide. Analysis of products by HPLC showed that the enzyme was selective in glycosylating peptides or glycopeptides with the PTTTPIST sequence in that the preferred primary glycosylation site was the third Thr from the aminoterminal end; secondary glycosylation depended on the site of the primary glycosylation. Negatively but not positively charged amino acids on the carboxy-terminal side of the putative secondary glycosylation site resulted in high activity suggesting charge-charge interactions of substrates with the enzyme. These studies indicate that O-glycosylation by bovine colostrum ppGalNAc-T is a selective process dependent on both the amino acid sequence and prior glycosylation of peptide substrates.Abbreviations Gal G,d-galactose - GalNac N-acetyl-d-galactosamine - HPLC high performance liquid chromatography - ppGalNAc-T UDP-GalNAc: polypeptide -GalNAc-transferase EC 2.4.1.41 - Ser^GalNAc GalNAc-Ser - Thr^GalNac GalNAc-Thr     

Synthesis of optically pure chrysobactin and immunoassay development

Chrysobactin (-N-(2,3-dihydroxybenzoyl)-d-lysyl-l-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of -N-(2,3-dibenzyloxybenzoyl)--N-Cbz-d-lysine with l-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10^–8 to 10^–10 mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.