Preparation and opioid activities of N-methylated analogs of [D-Ala2,Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala2,Leu5]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH3I-KHCO3 in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me3+Tyr1,D-Ala2,Leu5]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH3I-Ag2O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala2,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

Identification of gastrin releasing peptide-related substances in guinea pig and rat brain

Rat and guinea pig brain extracts were examined for the occurrence of gastrin-releasing peptide (GRP)-like substances by sequence specific radioimmunoassays interfaced with gel filtration and reversed phase high performance liquid chromatography (RP-HPLC). Tryptic digestion of the immunoreactive peptides followed by RP-HPLC was used to further characterize GRP-related peptides in brain. Using these analytical techniques it was found that guinea pig brain extracts contained a peptide with characteristics identical to authentic GRP (27 amino acid residues long). A carboxyterminal fragment with the characteristics of GRP(18–27) as well as a respective aminoterminal fragment with the characteristics of GRP(1–16) were also present in guinea pig brain extracts. The GRP(18–27) seems to correspond to the bombesin related material that has been described previously in mammalian brain extracts.Rat brain extracts also contained a peptide with the characteristics of GRP(18–27). The corresponding aminoterminal fragment, however, behaved differently on RP-HPLC from authentic GRP(1–16) and it was not recognized by antibodies directed to the aminoterminal tridecapeptide fragment of authentic GRP. Similarly the GRP-like peptide from rat brain did not comigrate on RP-HPLC with authentic GRP and was unreactive to antibodies directed toward the aminoterminus of GRP.     

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Immunohistochemistry of opioid peptides in the guinea pig endocrine pancreas

Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary.     

Tissue Content of Opioid Peptides in the Myenteric Plexus-Longitudinal Muscle of Guinea-Pig Small Intestine

We have developed a method that is based on two HPLC systems and permits the separation of endogenous opioid peptides in tissue extracts. The individual peptides are bioassayed on the mouse isolated vas deferens; naloxone (100 nM) ensures opioid specificity. In the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine, the tissue content of prodynorphin-derived peptides is lower than those of proenkephalin-derived peptides. No beta-endorphin was detected. Of the prodynorphin fragments, alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), and dynorphin B are present in equimolar concentrations (12-15 pmol/g) whereas the tissue content of dynorphin A is only 0.8 pmol/g. Processing of proenkephalin leads to at least six opioid peptides. The tissue contents of [Leu5]enkephalin, [Met5]enkephalyl-Arg-Gly-Leu, and [Met5]enkephalyl-Arg-Phe are 90-100 pmol/g and the content of [Met5]enkephalin is 405 pmol/g. BAM-18 and [Met5]enkephalyl-Arg-Arg-Val-NH2 are present in much lower concentrations, 24 and 5 pmol/g, respectively. Although present in low amounts, BAM-18 and [Met5]-enkephalyl-Arg-Arg-Val-NH2 have high affinity for the mu-opioid binding site and to a lesser extent for the kappa-site; this binding profile differs from that of the other proenkephalin fragments all of which have high affinities for the mu- and delta-sites.     

A general procedure for analysis of proenkephalin B derived opioid peptides

Tryptic digestion followed by radioimmunoassay for (Leu)enkephalin-Arg6 has been used in this study as a general method to detect the presence of all possible products containing the enkephalin sequence from the opioid peptide prohormone, proenkephalin B. Tissue extracts of human hypothalamus and pituitary were examined. Gel filtration was used to separate the different precursor products according to molecular weight. The elution profile was also monitored with highly sensitive radioimmunoassays for dynorphin A and dynorphin B, respectively. Immunoreactive dynorphin A appeared in three peaks with the approximate molecular weight of 1000, 2000 and 5000. Immunoreactive dynorphin B partly occurred in other peaks, 1500, 5000 and 10 000 dalton. Profiles obtained by measuring immunoreactive (Leu)enkephalin-Arg6 in all fractions from gel filtration after trypsin digestion showed a more complex pattern compared to the profiles of immunoreactive dynorphin A and dynorphin B. The major peaks coincided with dynorphin A and dynorphin B but high levels of immunoreactive (Leu)enkephalin-Arg6 were also generated from higher molecular weight regions (MW greater than 5000).     

Regional distribution of metorphamide in rat and guinea pig brain

A specific radioimmunoassay was developed for metorphamide, an endogenous, amidated opioid octapeptide, originally isolated from bovine brain and human pheochromocytoma tissues. The radioimmunoassay was used to determine the concentration of immunoreactive metorphamide in extracts from dissected regions of rat and guinea pig brain. Radioimmunoassay interfacing with Sephadex gel filtration and reverse phase high performance liquid chromatography confirmed that the immunoreactive substance measured corresponded to authentic metorphamide. Metorphamide was found to be widely distributed in brain regions from both species. However, the concentrations of immunoreactive metorphamide in regions from guinea pig brain were up to 5 times higher than the concentrations of immunoreactive metorphamide in rat brain regions. The results suggest that metorphamide is a specific processing product from proenkephalin in rodent brain.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Characterization of opioid peptides in the rat stomach

A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1-13. Closer examination of the dynorphin component revealed the presence of dynorphins 1-17, 1-13, and 1-8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.     

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain.

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.     

Identiflcation of a Proenkephalin Precursor in Striatal Tissue

Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin-containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin-containing intermediates do not accumulate. In this investigation Sephacryl-300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin-like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)-enkephalin, (Leu5)-enkephalin, and (Met5)-enkephalin-Arg6-Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)-enkephalin-Arg6-Phe7 constituted the C-terminal seven residues of this molecule.     

Synthesis and biological activity of [Leu5]enkephalin derivatives containing D-glucose

The synthesis of some [Leu5]enkephalin derivatives is described in which D-glucose has been linked to the opioid pentapeptide through the ester bond involving the carboxyl function at the C-terminal with C-1 or C-6 of the D-glucopyranose moiety. Enkephalin derivatives were assayed for opioid activity and found to be full agonists in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD). The obtained results suggest that the opioid activity of the tested glucoconjugates depend upon the ester bond position in the molecule. Whereas 1-O conjugate 5 was somewhat more potent than [Leu5]enkephalin in the GPI assay, the 6-O conjugates, with the exception of 1-O-benzyl derivative 11, were considerably less potent. All enkephalin derivatives were delta-receptor selective; in particular, the acetylated analog 8 was three times more delta-receptor selective than [Leu5]enkephalin.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.     

Met5-enkephalin-arg6-phe7 and its receptor in lung

The presence of met5-enkephalin-arg6-phe7 (YGGFMRF) and opiate receptors in rat, guinea pig and human lung was investigated with specific and sensitive radio immuno- and radio-receptor assays. 1) High and low molecular weight YGGFMRF-like immunoreactivity were detected in lung extracts using Bio-Gel P-2 column chromatography followed by radioimmunoassay. Using HPLC, we determined that the low molecular weight YGGFMRF-like immunoreactivity is authentic YGGFMRF. 2) The contents of YGGFMRF were 0.68 +/- 0.08, 0.76 +/- 0.12 and 0.63 pmol/mg protein in lung of rat, guinea pig and human, respectively. In the lung of these three species, the content of YGGFMRF is much greater than that of met5-enkephalin. 3) 47 mM KCl released YGGFMRF from rat lung slices in a Ca++ dependent manner. 4) Rat lung membranes were shown to bind [3H]-etorphine in a saturable manner. There are two populations of binding sites with a Kd = 0.6 and 7.1 nM and a Bmax = 7.8 and 28.5 fmol/mg protein, respectively. This binding could be displaced by YGGFMRF with high affinity, the other endogenous opioid peptides were poor displacers. From these results, we infer that YGGFMRF might be a putative neurotransmitter or neuromodulator, its role in the regulation of lung function can now be investigated.     

Focal Stimulation of the Mossy Fibers Releases Endogenous Dynorphins That Bind K1-Opioid Receptors in Guinea Pig Hippocampus

Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a kappa 1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid peptide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous kappa ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1-8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin- and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of kappa 1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that prodynorphin-derived opioids released under physiological conditions from the mossy fibers act at kappa 1 receptors in the guinea pig dentate gyrus.     

Glycoconjugates of opioid peptides. Synthesis and biological activity of [Leu5]enkephalin related glycoconjugates with amide type of linkage.

Three N-glycoconjugates of the general formula H-Tyr-Gly-Gly-Phe-Leu-NH-R (R = carbohydrate residue) were synthesized in order to determine the influence of some carbohydrate molecules (6-amino-6-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-glucopyranose, beta-D-glucopyranosylamine) on the biological activity, conformation, and stability of the opioid pentapeptide [Leu5]enkephalin. For the preparation of this compound different methods of peptide synthesis (active ester and mixed anhydride) were investigated. In comparison with [Leu5]enkephalin, all three N-glycoconjugates showed higher potency in the guinea pig ileum assay and lower potency in the mouse vas deferens assay, indicating a decrease in delta opioid receptor selectivity.     

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.     

Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.     

Detection of Met-enkephalin in the CNS of the teleosts, Anguilla rostrata and Oncorhynchus kisutch

Acid extracts of the brains of the American eel, Anguilla rostrata, and the coho salmon, Oncorhynchus kisutch, were screened for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography, a peak of Met-enkephalin-related immunoreactivity was detected near the total volume of the column for both species. No higher molecular weight forms of Met-enkephalin-related material were detected, nor were any immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) or alpha-neo-endorphin detected for either species. The enkephalin-sized immunoreactivity was further analyzed by reverse phase HPLC. For both species, a peak of authentic Met-enkephalin was detected. However, Leu-enkephalin, Met-enkephalin-RGL and Met-enkephalin-RF were not detected by RIA in either species. In addition, no novel C-terminally extended forms of Met-enkephalin were detected in either species. Finally, opiate receptor binding activity was only found associated with the peak of immunoreactive Met-enkephalin.