In order to immunoassay the specific region of bovine pituitary pro-opiomelanocortin (POMC) between ACTH and gamma-MSH, referred to as "joining peptide," antisera were prepared against the synthetic amidated decapeptide Val-Ala-Val-Gly-Glu-Gly-Pro-Gly-Pro-Arg-NH2. The non-amidated peptide represents residues -23 to -14 of bovine POMC. An NH2-terminal tyrosine analog of the decapeptide was used as the radioligand. Under optimal conditions, immunoassay with selected antisera exhibited a sensitivity (50% displacement of the radioligand) toward the decapeptide in the range of 31-55 pg. Immunoreactivity found in extracts of fresh or lyophilized bovine pituitary glands displaced the iodinated Tyr-decapeptide in the RIA in a parallel manner. The amount of immunoreactive (ir)-material was dependent upon the state of preservation of the tissue, the method of extraction, and the particular antiserum used. Extractable immunoreactivity was separated into low (Mr 1,500) and high (Mr 17,000) molecular weight peptides using gel chromatography (G-75). Additional ir-material appeared in the void volume (Mr greater than 22,500). Thus, these antisera have the capacity to interact not only with a region of the joining peptide but also with its larger, and apparent precursor forms. The immunoassay developed should be valuable in understanding the disposition and processing in this specific region of POMC.

Animals↗

PubMedDate not supplied

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment.

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.

Animals↗

PubMedDate not supplied

Interaction of deltorphin with opioid receptors: molecular determinants for affinity and selectivity.

Opioid receptor analyses of deltorphin A (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) analogues indicated the following: (a) increased negativity differentially affected affinities (Ki) and selectivity (Ki mu/Ki delta); (b) shifted sequence heptapeptides, [Asp5,Leu6,Met-NH2(7)] and [Asp4,His5,Leu6,Met-NH2(7)], reversed selectivity (delta-->mu); (c) substitutions at positions 4, 5, and 6 diminished selectivity, with changes in residue 5 being the most detrimental; (d) C-terminal deletions differentially effected Ki. These are the first data to demonstrate a reversal of delta selectivity in heptapeptides containing a negative charge and indicate that modifications in affinity occur through changes in both anionic and hydrophobic properties of residues at specific positions in the peptide. Deltorphin analogues might also be applied to differentiate between opioid receptor subsites.

Amino Acid Sequence↗

PubMedDate not supplied

Molecular dynamics conformations of deltorphin analogues advocate delta opioid binding site models.

Multi-site binding models for the delta opioid receptor were studied in vitro with [3H]DPDPE as the labeled ligand using analogues of deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) altered at position 4. Modifications included a change in chirality (L- to D-Asp4), increased length of the anionic side-chain (Glu4), elimination of the charged group (Abu4), addition of an anionic group (Gla4), and change in backbone conformation (Pro4). All of the peptides had relatively high delta affinities (0.09 to 1.15 nM); the major variability in delta selectivity resided in changes in mu affinities (1.6 to 530 nM). Three analogues (Glu4, D-Asp4 and Pro4) revealed better fits to two-site binding models (Hill coefficients < 0.850 with narrow 95% confidence intervals and P < 0.0001). Deltorphin C and analogues containing Gla4 and Abu4 (which were weakly delta selective), as well as deltorphin A (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2), fitted one-site binding models. Molecular dynamics simulations performed on deltorphin C and Abu4 exhibited similarities in the tertiary structure of their low energy conformers, while differing from the three-dimensional structures of the analogues containing Glu4, D-Asp4 and Pro4 substitutions. The data provide support that the three-dimensional architecture of an opioid peptide is an important factor in the designation of delta opioid receptor subtypes.

Amino Acid Sequence↗