Structure/activity relationships of C‐terminal neuropeptide Y peptide segments and analogues composed of sequence 1–4 linked to 25–36

Abstract

Abbreviations. ADPV, 4-aminomethyl-3,5-dimethoxyphenoxyvaleric acid; Ahx, 6-aminohexanoic acid; Aib, a-aminoisobutyric acid; Aoa, 8-aminooctanoic acid; BHA, benzhydrilamine; Aud, 11 -aminoundecanoic acid; Boc, butoxycarbonyl; tBu, t-butyl; Dns, dansyl; FAB-MS, fast-atom-bombardment mass spectrometry; ED, effective dose; Fmoc, fluoren-9-ylmethoxycdrbonyl; HOBtz, l-hydroxybenzotriazole; Hrg, homoarginine; IC, inhibitory concentration; iPr2C, diisopropylcarbodiimide; MeCI2, methylene chloride; Mtr, 4-methoxy-2,3,6-trimethylsulfonylbenzene; OSuc, N-hydroxysuccinimide ester. MATERIALS AND METHODS Thin-layer chromatographySolutions of peptides were applied on silica gel plates 60 F254 (Merck, Darmstadt). RF values were determined in solvent-saturated glass chambers at room temperature using the following solvent systems: A, methanol/chloroform/acetic acid (10:90:1); B, t-butanollwaterlacetic acid (2:2: 1); C, water/t-butanollacetic acid (3 : 4: 1); D, waterlt-butanollacetic acid (2:4: 1); E, chloroform/methanol/water (65:25:4); F, heptanelpyridine (7: 3), G, heptane/chloroform (3 : 7). The peptides were visualized by ultraviolet light (254 nm) and detected by spraying with water, ninhydrin and 4,4'-bis (dimethy1amino)diphenylmethane.

Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C‐terminal NPY analogues

“…Native NPY bound with the greatest affinity to these receptors ( Table 2). As previously shown, the centrally truncated analogue NPY(1 -4)-Ahx-(25 -36) binds with a similar affinity to NPY 19-36 and is 50 times more active than Ac-NPY(25 -36) ( Table 2) (Beck-Sickinger et al, 1990b). Binding was not altered by the exchange of Pro2 by His or Ile3' by Tyr.…”

“…Hydrophobic interactions of the proline helix and the amphilic ahelix lead to the hairpin structure. A model for the 3 dimensional structure of pNYP and hNPY has been deduced based on the X-ray structure of aPP (Allen et al, 1987;MacKerrell, 1988;Beck-Sickinger et al, 1990b). The hairpin structure was present in all molecular dynamics simulations.…”

Complete L‐Alanine Scan of Neuropeptide Y Reveals Ligands Binding to Y1 and Y2 Receptors with Distinguished Conformations

“…Starting from the X-ray structure of avian pancreatic polypeptide, a model has been deduced for the three-dimensional structure of porcine NPY (pNPY) and human NPY (hNPY) (Allen et al, 1987;MacKerell, 1988;. Beck-Sickinger et al, 1990~).…”

The Bioactive Conformation of Neuropeptide Y Analogues at the Human Y2‐Receptor

“…The resulting membranes were resuspended in 12.5 vol. of the above-mentioned Tris/HCl buffer containing 5 mM MgCI,, 0.1 mg/ml soybean trypsin inhibitor, 0.1 % serum albumin, and 0.25 mg/ml bacitracin (Beck-Sickinger et al, 1990b). For preparation of SMS-KAN and SK-N-MC cell membranes SMS-KAN cells were grown in 50% nutrient mixture Ham's F12/50% Dulbecco's modified Eagle's medium with 15% fetal calf serum, 2 mM glutamine, non-essential amino acids, 1 % gentamycin at 37°C and 5% CO, until they were confluent.…”

“…and different concentrations of the peptide in a total volume of 250 pl for 2 h at room temperature as described recently (BeckSickinger et al, 1994;Ingenhoven and Beck-Sickinger, 1997). For binding studies on rabbit kidney membranes, 230 p1 membrane solution containing about 100 pg protein were used to give a final volume of 240 p1 and incubation was carried out at 21 "C for 90 min (Beck-Sickinger et al, 1990b). Non-specific binding was defined in the presence of 1 pM NPY.…”