guanylyl-imidodiphosphate and dynorphin-(1-8)

Opioid binding sites specific for [3H]dynorphin1-8 were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphin1-8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin1-13, bremazocine, levorphanol and naloxone. The specific binding of [3H]dynorphin1-8 can be significantly inhibited by Na+ ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a Kd approximately 7.5 nM is present in the membranes. The maximum density of binding sites (Bmax approximately 1.1 pmol [3H]dynorphin1-8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the mu- and delta-types of opioid receptor showed that in the frog brain most of the sites labelled by [3H]dynorphin1-8 are kappa-sites and that this is a rich source of such sites.

Topics: Amino Acid Sequence; Animals; Brain; Cell Membrane; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Ethylketocyclazocine; Guanylyl Imidodiphosphate; Molecular Sequence Data; Naloxone; Peptide Fragments; Rana esculenta; Receptors, Opioid; Sodium Chloride; Tritium