enkephalin--leucine-2-alanine and dynorphin-amide-(1-10)

It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin(1-10)-Gly11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.

Topics: Affinity Labels; Amino Acid Chloromethyl Ketones; Analgesics, Opioid; Animals; Binding, Competitive; Brain Chemistry; Dihydromorphine; Dynorphins; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; Membrane Proteins; Peptide Fragments; Radioligand Assay; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Tritium

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain Chemistry; Chromatography, Gel; Cyclazocine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Membranes; Molecular Weight; Naloxone; Peptide Fragments; Pyrrolidines; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Solubility

The effectiveness of beta-endorphin, dynorphin-(1-13), dynorphin-(1-10) amide, alpha-neoendorphin and [D-Ala2,D-Leu5]enkephalin in suppressing withdrawal in heroin addicts was compared in this study. Groups of six patients were stabilized overnight in the hospital and were treated with either saline or peptide when withdrawal symptoms began to appear the following morning. Withdrawal was scored before and after treatment by the patient himself and an independent observer. Peptides were administered in a bolus dose of 60 micrograms/kg body weight. The patient, the observer and the physician who administered the injection were all blind to the nature of the compound given. All treatments, including those with saline, produced an overall reduction of withdrawal score. However, by statistical analysis, only treatments with beta-endorphin, [D-Ala2,D-Leu5]enkephalin and dynorphin-(1-13) were effective in producing a significant decrease of withdrawal symptoms. The length of relief brought about by the different peptides varied from less than an hour to a maximum of 5 h in one case. The average period of relief brought about by beta-endorphin, dynorphin-(1-13) and [D-Ala2,D-Leu5]enkephalin was 44, 46 and 60 min, respectively. Of the five peptides administered [D-Ala2,D-Leu5]enkephalin produced the largest number of side-effects.

Topics: Adult; Animals; beta-Endorphin; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Heroin; Humans; Male; Mice; Peptide Fragments; Protein Precursors; Substance Withdrawal Syndrome