Glucagon-like peptide-1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic p-cell. To characterize the side-chain groups required for interaction of glucagon-like peptide-1 with its receptor, we performed binding studies with alanine-substituted glucagon-like peptide-1 analogues on RINmSF insulinoma cells. The binding affinity and biological activity of glucagon-like peptide-1 have been found to be sensitive to alanine exchanges in the N-terminal positions 1, 4, 6 and the C-terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10-12, 14, 16-21 and 25-30 do not change receptor affinity. These findings could be exploited to design glucagon-like peptide-1 agonists and probably antagonists for further physiological studies.Glucagon-like peptide-1 is a 30-residue gastrointestinal hormone released from the enteroglucagon cells (L-cells) in the small intestine [l -31. The post-translational processing of proglucagon in the small intestine leads to the generation of glucagon-like peptide-1, corresponding to positions 78 -108 of the human proglucagon precursor [3, 41. In v i m , glucagon-like peptide-1 increases insulin secretion from the isolated rat [S] and pig pancreas [2, 61, and from isolated rat islets [7]. In man, glucagon-like peptide-1 is released into the circulation postprandially, and glucagon-like peptide-1 infusions stimulate insulin release [ 81. Therefore, glucagon-like peptide-1 plays an important role in the postprandial regulation of insulin secretion. Recent studies have shown that glucagon-like peptide-1 also has an anti-diabetogenic effect by reducing the isoglycaemic meal-related requirement for insulin in patients with non-insulin-dependent diabetes mellitus [9][10][11].Receptors for glucagon-like peptide-1 have been characterized in RINm5F cells [12-141, a Abbreviations. Fmoc, N-9-fluorenylmethoxycarbonyl; [HlAIglucagon-like peptide-1 , glucagon-like peptide-I , with an alanine exchange in position 1 for the naturally occurring histidine residue. All other glucagon-like peptide-1 analogues are named accordingly with the first letter giving the naturally occurring amino acid in the glucagon-like peptide-1 sequence and the number following the first letter giving the position of the exchanged amino acid.Note. This work is dedicated to Werner Creutzfeldt, Professor emeritus of Medicine, Georg-August University of Gottingen, Germany, on the occasion of his 70th birthday. the gene encoding the receptor has been localized in humans [17]. So far, little is known about the structural requirements for glucagon-like peptide-1 binding to its receptor. Binding studies with N-terminal and C-terminal glucagon-like peptide-1 fragments have shown that the C-terminal domains of the glucagon-like peptide-1 molecule are important for receptor binding. The N-terminal fragment glucagon-like peptide-1 (7 -2.5) did not show receptor binding, indicating that longer N-terminal fragments are needed for receptor recognition [ 1 31. The glucag...

A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.

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Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (Kd 0.23 nM, Bmax 44 fmol/mg), the hypothalamus (Kd 0.20 nM, Bmax 25 fmol/mg) and the cortex cerebri (Kd 0.11 nM, Bmax 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax < 3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125-I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.