Enzymatic transfer of sialic acids modified at C-5 employing four different sialyltransferases

We present kinetic studies on the enzymatic transfer of several synthetic sialic acid analogues, modified at C-5, to distinct glycoprotein glycans by sialytransferases differing in acceptor- and linkage-specificity. Biochemical properties of sialic acids were modified by introducing formyl-, trifluoroacetyl-, benzyloxycarbonyl-, and aminoacetyl-groups to the amino group at C-5 of neuraminic acid. The latter substitution renders the corresponding -glyocoside resistant towards sialidases. The respective CMP-sialic acid analogues were prepared by CMP-sialic acid synthase with a yield of 13–55%.The kinetic parameters of several sialyltransferases for the 5-substituted CMP-glycosides differed significantly. Relative to parent CMP-NeuAc, reaction rates of human- and rat liver Gal1, 4GlcNAc 2,6-sialyl-transferases ranged from 50 to 170%, of GalNAc 2,6-sialyltransferases from 40–140%, and of Gal1,3Gal-NAc 2,3-sialyltransferase from 20–50%. Resialylation of asialo-₁-acid glycoprotein by 5-N-formyl- and 5-N-aminoacetyl-neuraminic acid employing rat liver Gal1,4GlcNAc 2,6-sialyltransferase proceeded to about 80% of galactose sites which is identical to the extent achieved with parent NeuAc.According to our data, neosialoglycoconjugates which carry sialic acids modified at theN-acetyl group can be prepared for structure-function analysis, as this position seems crucial for recognition of adhesion proteins and influenza viruses.     

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.     

Constrained C-terminal hexapeptide neurotensin analogues containing a 3-oxoindolizidine skeleton

Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT_8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro¹⁰-Tyr¹¹ fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT_8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala¹¹]-NT_8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe¹¹]NT_8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT_8–13 analogues and [Phe¹¹]NT_8–13 or NT_8–13, it is not clear whether the -turn around Pro¹⁰-AA¹¹ (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro¹⁰-Tyr¹¹ dipeptide in NT_8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors.     

Somatostatin analogues containing o-aminomethylphenylacetic acid as a bridge unit

Summary A new cis-peptide bond mimetic, -benzyl-o-aminomethylphenylacetic acid, was synthesized and incorporated in a homodetic somatostatin analogue. Biological binding tests and 2D NMR conformational analysis indicate that the configuration of the bridge-unit asymmetric center and the orientation of the benzyl side chain play a key role in the biological activity of this type of somatostatin analogues.     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Inhibition of yeast tRNATrp aminoacylation by 4-methyltryptophan

The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNA^Trp). The mode of inhibition is competitive and the analogue is not charged onto tRNA^Trp. Thus 4-methyltryptophan application depletes the cells from charged tRNA^Trp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.     

Antibacterial and antimycotic effect of a newly discovered secretion from larvae of an endoparasitic insect,Pimpla turionellae L. (Hym.)

Three analogues of the peptidyl pheromone, pheromone of Saccharomyces kluyveri, synthesized based on the amino acid sequence proposed by Sato et al. (Agric Biol Chem 45:1531–1533, 1981) were tested for both shmoo-inducing and agglutinability-inducing actions. Purified natural pheromone of the yeast showed the highest activity among the peptides tested. When methionine in the peptides was oxidized, the activity decreased significatly. Pheromone of S. kluyveri induced sexual agglutinability in a cells of Saccharomyces cerevisiae, and shmoo in a cells of S. cerevisiae and S. kluyveri. a Pheromone of S. kluyveri had no agglutinability-inducing action on cells of S. cerevisiae. a Cells of S. kluyveri inactivated only pheromone of the same species, but a cells of S. cerevisiae inactivated pheromones of both S. cerevisiae and S. kluyveri.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Influx of γ-Aminobutyric Acid and α-Aminoisobutyric Acid into Mouse Cerebrum Slices Incubated in a Pyruvate Medium with or without Added Glucose or Glucose Analogues Compared with Influx from a Glucose Medium

Concentrative influx of γ-aminobutyric acid (GABA) and α-aminoisobutyric acid (AIB) into incubated mouse cerebrum slices is decreased when pyruvate is substituted for glucose. Influx of GABA from pyruvate medium is not increased by presence of glucose, 2-deoxy-d -glucose (2-DOG), or 3-O-methyl-d -glucose (3-O-MeG). Influx of AIB is restored to the rate from glucose medium if 2-DOG is present initially, but is not restored if 2-DOG is added with AIB. Influx is not restored if 3-O-MeG is present initially, but is restored if 3-O-MeG is added with AIB. Influx is restored if glucose is present initially or is added with AIB.