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The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at −70 °C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,AT-methyl-Phe4,Gly-ol5][3H] enkephalin for µ-receptors and [D-Ala2,d-Leu5][3H]enkephalin for δ-receptors. δ-Receptor binding assays were conducted in the presence of 2.6 µM[N-Me-Phe3, D-Pro4]morphiceptin to suppress peptide binding to µ-receptors. [D-Ala2, N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nMfor receptors and 355.8 and 178.6 nMfor δ-receptors, respectively.

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.

Two peptides exhibiting kinin activity in an isolated rat uterus assay were purified from pasteurized skim bovine milk. The amino acid sequence of the more prominent peptide was found to be that of bradykinin. Partially purified kinin preparations were also obtained from N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin digests of non-fat dry milk and insoluble lactalbumin. The application of fast atom bombardment/mass spectrometry permitted detection of the bradykinin protonated molecular ion in each of these samples. Collision-activated decomposition of the ion of m/z1061 confirmed it to be the protonated molecular ion of bradykinin. Fast atom bombardment/mass spectrometry analysis further confirmed the occurrence of bradykinin in a pancreatic kallikrein digest of a partially purified bovine milk kininogen preparation.

Skin of the frog Phyllomedusa sauvageicontains a cDNA sequence that codes for the selective μ-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide(DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for δ-type opioid receptors in rat brain synaptosomes. The IC50values for DGAP on μ- and δ-receptors were only 28 µMand 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nMfor δ-receptors and > 1 µMfor μ-receptors yielding a very high δ selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2, D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for δ-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2residue and is a selective μ-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem.264, 354–362), exhibits a SR of 0.0055 similar to that for the conventional μ-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for μ- and δ-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.