Lipidation with long-chain di-fattyacyl-glycerol moieties was used to anchor gastrin in CCK peptides irreversibly to lipid bilayers lipopeptide transfer to model phospholipid bilayers is fast and quantitative, leading to a different mode of insertion of lipo-gastrin and lipo-CCK in lipid layers. Lipo-gastrin remains exposed to the bulk solvent in a predominantly random coil structure as a consequence of electrostatic repulsion, whereas lipo-CCK exhibits a pronounced tendency to form peptide domains with insertion of its C-terminus into more hydrophobic compartments of the bilayers. Thereby Ca2+ at physiological concentrations favours this aggregational phenomenon. Since both lipo-peptides were found to retain almost full receptor affinity despite their irreversible anchorage to the bilayer, a membrane-bound pathway in the receptor recognition and binding process is indeed possible. According to the data collected in this study, CCK might possibly use this pathway, whereas accumulation of gastrin on the cell membrane with prefolding of the ligand at the water/lipid interface is hardly conceivable. Nevertheless the observed receptor interaction of the deliberately membrane-anchored gastrin offers interesting constraints for computational docking experiments on a modelled CCK-B/gastrin receptor by additionally taking into account information derived from mutagenesis studies. Despite the limitations of such modelling experiments, the resulting picture of the gastrin/receptor complex allowed the visualization and rationalization of the experimental results of the extensive structure-function studies performed previously on this family of gastrointestinal hormones.

Animals↗

PubMedDate not supplied

Synthesis of selenocysteine peptides and their oxidation to diselenide-bridged compounds.

Using the Fmoc/tBu protection scheme and the p-methoxybenzyl derivative of selenocysteine, the synthesis of related peptides in the selenol-protected form could be optimized by operating the coupling steps in the absence of auxiliary bases and by reducing the piperidine treatment to the minimum time required for quantitative Fmoc cleavage. Under these conditions, beta-elimination of the p-methoxybenzylselenol as the main side reaction of these syntheses, as well as epimerization of the protected selenocysteine, was largely suppressed. Conversion of the selenol- and thiol-protected bis-selenocysteine and selenocysteine, cysteine peptides into the related cyclic monomeric forms by iodine-mediated oxidation failed since a complex mixture of compounds was produced. Cleavage of the selenoether bond with mercuric acetate was found to proceed smoothly, but displacement of the heavy metal ions by treatment with excesses of thiols or hydrogen sulphide was unsuccessful since a stable Hg2+ diselenide complex was obtained. However, oxidation was achieved in good yields by the dimethylsulphoxide/trifluoroacetic acid procedure and the peptides were then used for determining the redox potential of the diselenide and selenide/sulphide bridge, respectively.

Organoselenium Compounds↗

PubMedDate not supplied

Peptide/benzodiazepine hybrids as ligands of CCK(A) and CCK(B) receptors.

The (neuro)hormones gastrin and cholecystokinin (CCK) share a common C-terminal tetrapeptide amide sequence that has been recognized as the message portion while the N-terminal extensions are responsible for the CCK(A) and CCK(B) receptor subtype selectivity and avidity. 1,4-Benzodiazepine derivatives are potent and selective antagonists of these receptors, and according to comparative molecular field analysis, the structures of these nonpeptidic compounds could well mimic the message sequence of the peptide agonists at least in terms of spatial array of the aromatic residues. Docking of a larger series of low molecular weight nonpeptide antagonists to a homology modeling derived CCK(B) receptor structure revealed a consensus binding mode that is further validated by data from site-directed mutagenesis studies of the receptors. Whether this putative binding pocket of the nonpeptide antagonists is identical to that of the message portion of the peptide agonists, or whether it is distinct and spatially separated, or overlapping, but with distinct interaction sites, is still object of debate. Using a 1,4-benzodiazepine core amino-functionalized at the C3 position, related tryptophanyl derivatives were synthesized as mimics of the tetrapeptide and subsequently extended N-terminally with gastrin and CCK address sequences. All hybrid constructs were recognized as antagonists by the CCK(A) and CCK(B) receptors, but their address portions were incapable of enhancing in significant manner selectivity and avidity. Consequently, the binding of the peptide/benzodiazepine hybrids has to be dictated mainly by the benzodiazepine moiety, which apparently prevents optimal interactions of the address peptides with extracellular receptor subdomains. These findings would strongly support the view of distinct binding sites for the message portion of the peptide agonists and the benzodiazepine-based nonpeptide antagonists.

Benzodiazepines↗

PubMedDate not supplied

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments.

The fully active gastrin and CCK analogues [Nle 15]-gastrin-17 and [Nle, Thr]-CCK-9 were analysed for their Ca2+ and Tb3+ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca2+ and Tb3+, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to beta-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca2+ concentrations. Induced lipid interaction via N-terminal lipo-derivatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca2+ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca2+ enhances the clustering of the peptide headgroups in a more beta-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca2+ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca2+ for the peptides could well play a role in the inhibitory activity of lanthanum on the signal transduction of the CCK family of hormones.

Amino Acid Sequence↗

PubMedDate not supplied

Cysteine racemization in peptide synthesis: a new and easy detection method.

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris-alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine-peptides. Baseline enantiomeric resolution of the D,L-S-beta-(4-pyridylethyl)cysteine, and thus quantification of D-enantiomer contents at levels < or = 1%, is easily achieved by capillary zone electrophoresis exploiting the host-guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.

Amino Acid Sequence↗

PubMedDate not supplied

Role of idiotype-independent anti-IgG autoantibodies in human kidney transplantation: natural anti-F(ab')2 antibodies recognize an IgG1 hinge region epitope.

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Amino Acid Sequence↗