Peptide research最新文献

The possibility that hydrophobic interactions may be used as a basis for the design of receptor mimetic peptides for small peptide hormones that lack the potential to adopt amphiphilic secondary structures was tested by designing and characterizing receptor mimetic peptides for gamma-endorphin. The receptor mimetic peptides were designed to exhibit a pattern of hydrophobic surfaces in an antiparallel orientation matching that of the peptide hormone in an extended conformation. An ELISA-based assay was used to determine the relative binding affinities of receptor mimetic peptides, control peptides and antisense peptides to gamma-endorphin immobilized on a surface. The inhibition constant for the best gamma-endorphin receptor mimetic peptide was 1.6 microM. No binding was detected for scrambled control peptides or the antisense-derived peptide mimetic to the limit of their respective solubilities. Sera from rabbits immunized with a gamma-endorphin receptor mimetic peptide were used to immunopurify the ligand-binding domain of the human opiate receptor and were cross-reactive with purified bovine opiate receptor. These results suggest that patterns of hydrophobicity can provide a rational basis for designing receptor mimetic peptides and may provide an explanation for the ability of some antisense peptides to bind to their cognate hormones and to elicit antibodies cross-reactive with hormone receptors.

Recently, we constructed a series of highly efficient universal eukaryotic gene expression vectors (Sheay et al., BioTechniques 15:856-862, 1993). Such vectors contain a viral promoter and enhancer followed by the adenovirus tripartite leader sequence, a multiple cloning site for the insertion of the gene of interest and a polyadenylation sequence. To enable expression of peptides to be secreted into the tissue culture medium or to be incorporated into the cell membrane, several modifications have been introduced into such vectors: stop codons in all three reading frames were inserted at the end of the multiple cloning site and a DNA sequence coding for a signal peptide for transport through the endoplasmatic reticulum (ER) was introduced downstream of the adenovirus tripartite leader sequence followed by two unique restriction enzyme recognition sites. A protocol is described that allows fast and easy cloning of peptide-coding regions, i.e., PCR products, for expression and secretion. The transport of a genetically engineered chimeric transmembrane protein connected to this ER leader sequence was as efficient as that of the original protein from which the ER sequence has been derived. These universal vectors can also be used for the easy construction of any chimeric transmembrane or secretion proteins.

The suprachiasmatic nucleus (SCN) receives a direct photic projection from the retina, the retinohypothalamic tract (RHT), and an indirect photic projection from the intergeniculate leaflet of the thalamus, the geniculohypothalamic tract (GHT). The primary neurochemical signal in the GHT appears to be neuropeptide Y (NPY), and several lines of evidence indicate that NPY may be involved in determining the response of the SCN to light. The purpose of the present study was (i) to localize NPY binding sites in the hamster SCN and to compare the distribution of these binding sites with the terminal field of the RHT and (ii) to determine if SCN levels of NPY binding change during the day. RHT fibers, defined using the carbocyanine dye DiI, were localized primarily within the ventrolateral region of the SCN. The distribution of NPY receptors, as visualized by 125I-peptide YY (PYY) binding, overlapped the projection field of the RHT. Specific binding of 125I-PYY was significantly greater in the ventrolateral SCN than in the medial SCN. To determine whether NPY binding changes during the day, the levels of 125I-PYY in the SCN were determined 4 h before the onset of darkness, 1 h before the onset of darkness and 2 h after the onset of darkness in hamsters housed in a 14:10 light-dark cycle. The levels of binding at 4 and 1 h before dark onset were significantly lower than 2 h after the onset of darkness. In contrast, no significant differences were observed in 125I-PYY binding over these same sampling intervals in either the medial preoptic area or the lateral hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)

The conformation of a synthetic peptide corresponding to residues 35-53 (SAKM-REWFSETFQKVKEKL) of human apolipoprotein C-I (57 amino acids) was studied by nuclear magnetic resonance and circular dichroism spectroscopy in water and in perdeuterated dodecylphosphocholine solution at 37 degrees C and pH 4.8. The proton resonances of the peptide in both solutions were assigned from TOCSY, NOESY and DQF-COSY experiments. In water solution, the peptide is predominantly "random", although nuclear Overhauser connectivity patterns and H alpha secondary shifts show a threshold population of nascent helical conformers. Upon the addition of 40-fold molar excess dodecylphosphocholine to the water solution, the peptide adopts a helical structure that extends throughout the sequence.

Oxazolones and symmetrical anhydrides react efficiently with amino acids if the aqueous solvent includes a high proportion of dimethylformamide, the temperature is not lowered and sodium carbonate is used for neutralizing the amino acids. Subsequent separation of the diastereomeric products by high-performance liquid chromatography on a C-18 column provides a simple method for determining enantiomers. The symmetrical anhydrides of N-ethoxycarbonylvaline and phenyl-alanine are reagents suitable for determining enantiomers. Enantiomers of arginine and histidine in a mixture of amino acids can be determined if the products of the reaction are first fractionated by a simple extraction.

Peptide 1, cyclo(1,5-epsilon-succinoyl) (Lys-Gly-Gly-Gly)2, is a representative member of a family of polycyclic peptide ionophores characterized by C2 symmetry and a relatively flexible structure resulting from its high Gly content. Peptide 1 has been synthesized by two different solid-phase protocols from its linear precursors, H-Gly-Gly-Lys(Fmoc)-Gly-Gly-Gly-Lys(Fmoc)-Gly-OH and H-Gly-Gly-Lys(Z)-Gly-Gly-Gly-Lys(Z)-Gly-OH), and satisfactorily characterized by chemical means. The CD spectrum of 1 is compatible with a beta-folded structure, stabilized by two internal hydrogen bonds. The complexation behavior of 1 toward alkaline and alkaline-earth cations can be envisaged as an equilibrium between inclusion (1:1) and sandwich (2:1) complex models, with affinities in the 10(6) M-1 and 10(11) M-2 range, respectively. A slight preference of 1 for Sr2+ over other cations has been found.

The dipeptide Mtr-Asp-D-Adf-Pip 10 represents a potent thrombin inhibitor. In comparison to NAPAP, 10 exhibited improved tolerability and a longer half-life in vivo, i.e., 20 +/- 5 min. We have coupled aminopolyethyleneglycolmonomethylether of various molecular weights to the carboxyl moiety of 10 and evaluated their biological properties. First, Mtr-Asp-OBut was coupled to the amino group of the PEG employing TOTU as an activating agent. This was followed by the removal of the OBut protecting group and coupling of D-Adf-Pip using TOTU as well. The PEG-bound thrombin inhibitors showed inhibition constants vs. thrombin in the subnanomolar range, i.e., they were more active than the parent molecule 10. Moreover, the pegylated inhibitors exhibited a longer lasting effect in vivo. In rats the half-life of Mtr-Asn (PEG10000-OMe)-D-Adf-Pip 14 was determined to be 63 min. Mtr-Asn(PEG10000-OMe)-D-Adf-Pip 14 showed a half-life of 120 min in pigs. It could be concluded that these PEG-bound thrombin inhibitors may be employed as versatile drugs for parenteral administration in treating thrombotic disorders.

The multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has allowed synthesis at scales up to 10 mumol per pin. At this loading, the methodology can no longer be considered just a screening tool. The overall synthesis efficiency of this approach was assessed by the synthesis of 2913 different peptides having little or no sequence homology and ranging up to a 46-mer in length. High performance liquid chromatography analysis of the crude peptides indicates overall quality of synthesis was high. The method is suitable for multi-milligram synthesis of peptides without sacrificing any of the inherent advantages of the 96-well format.

We recently proposed a model of bradykinin bound to the rat bradykinin B2 receptor that is constructed on the basis of structural homology modeling to the criomicroscopic structure of the seven transmembrane domains of bacteriorhodopsin, extensive conformational searches and experimental mutagenesis results. On the basis of that model, a novel third-generation pseudopeptide antagonist, NPC 18325 (D-Arg1-Arg2-[aminotridecanoyl]3-Ser4-D-Tic5-Oic6++ +-Arg7) (Ki = 440 nM, guinea pig ileum), was designed and also reported. NPC 18325 has been proposed to adopt a C-terminal beta turn separated from N-terminal positive charges by a linear 12 carbon chain spacer. Experimentally, the four amino acids making up the C-terminus have been shown by NMR to preferentially adopt a beta turn at neutral pH in aqueous solution. We now present a series of peptides, related to and including NPC 18325, that explore the relationship between the length of the carbon chain and the affinity to the human bradykinin B2 receptor. The results show that there is a structure-activity relationship (SAR) associated with the chain length and that these pseudopeptides have better affinity to the human bradykinin receptor than they have to the guinea pig ileal-derived B2 receptor. Specifically, peptide I (a 12-methylene linker) had a measured Ki of 31 nM and peptide V (a 4-methylene linker) had a Ki of 471 nM. Implications regarding conformation and hydrophobicity are also described.