Conformational analysis of bioactive peptides in reverse micelles as mimics of cell membrane environments

Summary Conformational preferences of secretin as a model peptide have been analyzed by CD and IR spectroscopy in reverse micelles of AOT/isooctane/water and compared to those in aqueous TFE, in SDS micelles and in DMPG vesicles. Among the systems examined, reverse micelles and phospholipid vesicles displayed almost identical conformational equilibria. Very high lipid-to-peptide ratios can be obtained in reverse micelles with full retention of optical transparency, even at millimolar peptide concentrations, thus indicating this system to be an interesting mimic of cell membrane environments for spectroscopic analysis of bioactive peptide conformations.Abbreviations TFE trifluoroethanol - SDS sodium dodecyl sulfate - DMPG dimyristoylphosphatidylglycerol - AOT bis(2-ethylhexyl)sulfosuccinate - CMC critical micellar concentration - VIP vasoactive intestinal peptide     

Conformational studies of chemotactic HCO-Met-Leu-Phe-OMe analogues

Summary In order to investigate the proper peptide backbone conformation able to elicit a biological activity, HCO-Met-Pro-Phe-OMe, HCO-Met-[COO]Leu-Phe-OMe, and HCO-Met-OLeu-Phe-OMe, analogues of the prototypical chemotactic peptide HCO-Met-Leu-Phe-OMe, were studied by CD and IR techniques. The results obtained comparing biological and conformational data evidence the critical presence of (i) the NH group at position 2, (ii) a rather flexible backbone, (iii) the chemical structure of the central residue which can affect the stability of a possible active conformer.     

Demonstration of high opioid-like activity in isolated peptides from wheat gluten hydrolysates

Because of a possible relationship between schizophrenia and celiac disease, a condition in some individuals who are sensitive to wheat gluten proteins in the diet, there has been interest in observations that peptides derived from wheat gluten proteins exhibit opioid-like activity in in vitro tests. To determine the origin of the peptides exhibiting opioid activity, wheat proteins were fractionated by size (gel filtration), by charge differences (ion exchange chromatography) and by differences in hydrophobicity (reversed-phase HPLC). These fractions were hydrolyzed by pepsin or pepsin and trypsin and the resulting peptides separated by gel filtration chromatography. The separated peptides were tested for opioid-like activity by competitive binding to opioid receptor sites in rat brain tissue in the presence of tritium-labeled dihydromorphine. The peptides showed considerable differences in activity; while some peptides exhibited no activity, 0.5 mg of the most active peptides were equivalent to 1 nM of morphine in the binding assay. The most active peptides were derived from the gliadin fraction of the gluten complex.     

High-performance liquid chromatography of iodine-labeled insulin and glucagon derivatives with on-line γ-detection

Insulin and glucagon were labeled with iodine. The reaction products were analyzed by high-performance liquid chromatography. It is shown that the pH of the reaction medium has a large effect on the position and the degree of iodine substitution as well as on the oxidation of the Met-containing glucagon and, furthermore, that the molar ratio of iodine to polypeptide hormone used during the labeling procedure affects not only the amount of iodine incorporated but also the distribution of iodinated products. The results show that certain iodinated derivatives are separated from each other and from the respective unlabeled polypeptide and thus can be obtained in a pure state.     

An apparatus for simultaneous manual solid-phase synthesis of multiple peptide analogs

A new design of reaction vessel for simultaneous manual solid-phase synthesis of multiple peptide analogs is described. Simultaneous use of four of these vessels attached to a single rotary mixer has been successfully applied to synthesis of two sets of four decapeptide amide analogs. Efficient coupling was indicated by chemical determination at the end of each synthesis cycle and overall final yields of between 78 and 84% were obtained. The products obtained were of a high quality, as assessed by high-performance liquid chromatography and amino acid analysis. This system allows expeditious synthesis of multiple peptide analogs for structure-function studies with economical use of efficiently ventilated laboratory space.     

I. Photoaffinity probes provide a general method to prepare synthetic peptide-conjugates

A general synthetic strategy is described for the preparation of peptide-conjugates where the peptides contain the NH₂ terminal, COOH terminal, or internal regions of the protein sequence. Glycoprotein D of herpes simplex virus type 1 is used as a representative protein. Ten-residue peptide fragments of the native sequence were synthesized using standard solid-phase methodology. Photoprobes stable to conditions of synthesis and HF cleavage were coupled directly to the protected-peptide resin during synthesis. This one-step procedure eliminates the potential modification of functional groups in the sequence of interest that can occur when using chemically labile bifunctional reagents. Since the photoprobe is inert until photolysis, the synthetic peptide-probe can be readily purified by high-performance liquid chromatography before cross-linking to the carrier molecule. The following photoprobe derivatives were investigated: thep-azidobenzoyl,p-nitrophenylalanyl, andp-benzoylbenzoyl groups. The benzophenone photoprobes were shown to give the highest incorporation of peptide-probe with the protein carrier over a wide range ofpH and solvent conditions. For solid-phase synthesis three benzophenone photoprobes can be used: benzoylbenzoic acid, benzoylbenzoylglycine, andN ^e-(4-benzoylbenzoyl)-N -t-butyloxycarbonyl-lysine.     

Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera)

Summary The distribution of gastrin/CCK-like immunoreactive material has been studied in the retrocerebral complex of Calliphora. The material reacts with antisera specific for the common COOH terminus of gastrin and CCK but not with N-terminal antisera. The three thoracic ganglia and the fused abdominal ganglia each contain a specific number of symmetrically arranged immunoreactive cells both dorsally and ventrally in pairs on either side of the midline in a sagittal plane. The neuropil of these ganglia also contains a considerable amount of immunoreactive fibres and droplets. Reconstructed axonal pathways suggest that some of the nerve fibres have their origins within the brain and/or the suboesophageal ganglion. Immunoreactive material may also be seen apparently leaving the thoracic ganglion posteriorly via the abdominal nerves, and there is strong evidence of a neurohaemal organ within the dorsal sheath in the region of the metathoracic and abdominal ganglia. There appears to be a direct correlation between the content of peptidergic material of cells and fibres and the age and diet of the flies. The corpus cardiacum contains COOH-terminal specific gastrin/CCK-like material within the intrinsic cells and in the neuropil. It is present also in the cardiac-recurrent nerve entering the corpus cardiacum anteriorly and in the nerves leaving the gland dorsoposteriorly, the aortic or cardiac nerves. It is not observed, however, in the nerves leaving the corpus cardiacum ventroposteriorly, the so-called oesophageal, gastric or crop-duct nerves. The corpus allatum and the hypocerebral ganglion do not contain immunoreactive material of this type. Gastrin/CCK-like and secretin-like immunoreactive materials appear to co-exist in the cells of the corpus cardiacum and co-existence of gastrin/CCK-like and pancreatic polypeptide like substances occurs within certain cells of the thoracic ganglion.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Secretion of melanophore-stimulating hormone (MSH) in long-term cultures of pituitary neurointermediate lobes

Summary Neurointermediate lobes from pituitaries of the frog, Rana berlandieri forreri (Rana pipiens, sensu lato), were maintained in organ culture in media with and without serum for up to six months. The cultured tissues were examined periodically by light microscopy and transmission electron microscopy and by bioassay of the melanophore-stimulating hormone (MSH) secreted and present in the culture media. Light-microscopic observations revealed a high degree of preservation of the pars intermedia at four weeks with isolated areas of some glands maintaining histological integrity for the entire six months. Similarly, at the ultrastructural level the cells appeared morphologically intact and to be actively synthesizing and secreting hormone. Bioassays showed the glands to be continuously secreting MSH; however, larger yields of hormone were obtained in media lacking serum. No significant ultrastructural differences between cells grown in the presence or absence of serum were detected. The difference in concentration of MSH between the two groups therefore apparently results from enzymatic degradation of the hormone by the serum. Organ culture of the vertebrate neurointermediate lobe may provide a unique method for the production of large quantities of MSH and for the study of other melanotropic and opiate peptides as they may be synthesized and secreted by the pars intermedia.     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.