Nuclear magnetic resonance study of side-chain conformation of phenylalanine residue in [Met5]-enkephalin: solvent, pH, and temperature dependence.

[Met5]-Enkephalin and N-acetylphenylalanine methylamide containing (2S,3S)-[2,3-2H2]Phe were synthesized 270 MHz 1H NMR spectra of the normal and selectively deuterated species were analysed. The lower-field and higher-field beta-proton signals of the Phe4 residue of [Met5]-enkephalin were unambiguously assigned to the pro-S and pro-R protons, respectively. The same assignments apply to N-acetylphenylalanine methylamide in polar organic solvents and in 2H2O, but the alternative assignments apply in C2HCl3. For [Met5]-enkephalin, the vicinal spin coupling constants 3JalphabetaS and 3 JalphabetaR and the rotamer populations around the Calpha-Cbeta bond were determined in a variety of solvents. From the pH and temperature dependences of rotamer populations of [Met5]-enkephalin, the side-chain conformation of the Phe residue in 2H2O solution was found to be considerably different from that in (C2H3)2SO solution. Rotamer populations of the Phe4 residue of [Met5]-enkephalin in organic solvents depend on solvent polarity. As compared with the reference model molecule of N-acetylphenylalanine methylamide, the rotamer populations of Phe4 of [Met5]-enkephalin are affected possibly by steric repulsion with other residues; the rotamer I is primarily favored but the rotamer II is appreciably destabilized in weakly polar solvents.     

Naltrexone-sensitive behavioral actions of the ACTH 4-9 analog (Org 2766)

The effects of the ACTH 4-9 analog (Org 2766) and the COOH-terminal tripeptide of Org 2766 (Phe-D-Lys-Phe; PDLP) on retrieval of one-trial learning passive avoidance behavior were compared with those of beta-endorphin, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin, des-Tyr1-[Met5]-enkephalin and des-enkephalin-gamma-endorphin (DE gamma E). Amounts of intracerebroventricularly administered Org 2766, PDLP, [Met5]-enkephalin, [D-Ala2,Met5]-enkephalin and DE gamma E, which induced a comparable attenuation of passive avoidance behavior were determined. Pretreatment with the opiate antagonist naltrexone prevented the attenuating effect of these peptides on passive avoidance behavior except that of DE gamma E. The attenuating effect of Org 2766 and of [Met5]-enkephalin was reversed to facilitation of passive avoidance behavior in the presence of naltrexone. Subcutaneous treatment with Org 2766 and [D-Phe7]-ACTH 4-10 decreased electrical self-stimulation behavior elicited from the medial septal area. Naltrexone prevented the inhibitory effect of Org 2766 on this behavior, but not that of [D-Phe7]-ACTH 4-10. Although the attenuating effect of PDLP on passive avoidance behavior was not reduced by pretreatment with [Met5]-enkephalin- or beta-endorphin-antiserum, and PDLP induced neither analgesia nor excessive grooming, the data suggest that the inhibitory effect of Org 2766 and PDLP on passive avoidance behavior and electrical self-stimulation are mediated by endorphin systems in the brain.     

Conformational states of Leu5- and Met5-enkephalins in solution

The molecular conformations of Leu5- and Met5-enkephalins in aqueous and DMSO solutions were investigated by FT-IR and laser Raman spectroscopic methods. The amide I, II, and III regions in the FT-IR spectra of Leu5- and Met5-enkephalins in aqueous solution were analyzed by performing Fourier self-deconvolution of the bands. Leu5-enkephalin in aqueous solution is found to exist in both type II beta-turn and beta-sheet structures, whereas Met5-enkephalin has a lesser tendency to form beta-turn structure in aqueous solution. It is likely that these different conformers of enkephalins might bind to different receptor types.     

Endogenous opioids and the growth regulation of a neural tumor

Endogenous opioid systems (endogenous opioids and their receptors) are known to participate in the regulation of tumor growth. The present study was conducted to examine whether [Met5]-enkephalin influences the growth of transplanted neuroblastoma, and to explore the role of other opioid peptides in carcinogenesis. A/Jax mice were inoculated with 10(6) S20Y cells and received daily injections of [Met5]-enkephalin. Dosages of 0.5 to 30 mg/kg delayed tumor appearance and prolonged survival of these mice; antitumor effects were blocked by concomitant injections of naloxone. Daily administration (10 mg/kg) of [Leu5]-enkephalin had no effect on neurotumor growth. [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine, ligands selective for delta and kappa receptors, respectively, also did not influence neuro-oncogenesis. These results demonstrated the potent growth inhibiting effects of the naturally occurring opioid pentapeptide, [Met5]-enkephalin, and substantiate reports identifying and characterizing an opioid receptor (i.e., zeta) for which [Met5]-enkephalin is the most potent ligand.     

Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.     

Semicarbazide Substitution Enhances Enkephalins Resistance to Ace Induced Hydrolysis

In our previous study on [Met5]-enkephalin analogues, [Met5]-enkephalin semicarbazide was found as a new enkephalin amide that produces antinociception even in ACE (Angiotensin Converting Enzyme) exposure in vivo. In the present work we examined the corresponding [Leu5]-enkephalin derivatives to confirm the influence of semicarbazide substitution. To prevent the enkephalins biodegradation animals were pretreated with a mixture of peptidase inhibitors. As assessed by tail-flick test no significant difference was detected between the produced antinociception by the [Leu5]-enkephalin derivatives. Based on our results both semicarbazide and ethylamide groups could preserve the provided analgesia after captopril (ACE inhibitor) omission from the peptidase inhibitors mixture. This work confirms that semicarbazide substitution on enkephalins yields ACE resistance antinociceptive peptides, nevertheless it may necessarily not enhance the peptides analgesic potencies.     

Solution and membrane structure of enkephalins as studied by infrared spectroscopy

The backbone conformation of the two opioid pentapeptides Leu5-enkephalin and Met5-enkephalin was studied by the technique of resolution-enhanced infrared spectroscopy. In aqueous solution, the conformation-sensitive amide I bands of the two peptides are identical. The positions of these bands are consistent with the view that in aqueous solution both enkephalins exist as an ensemble of largely unfolded conformers. Interaction of Leu5- and Met5-enkephalins with bilayer membranes of ditetradecylphosphatidylcholine results in a substantial refolding of the peptide backbones. The conformation stabilized by the membrane environment is a hydrogen-bonded turn structure. Conformational transitions in enkephalins induced by a lipid environment may play a role in the specific interactions between these hormones and their receptor sites.     

Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.     

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.     

Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Nuclear Overhauser effects in linear peptides. A low-temperature 500 MHz study of Met-enkephalin

Met5-enkephalin was studied in 1 mM solutions in 2H2O at room temperature and in a cryoprotective mixture (DMSOd6/2H2O, mole fraction of DMSO 0.49) in the temperature range 265-298 K. Small positive effects were observed between the ortho and meta protons of Tyr in aqueous solution at room temperature. Intraresidue effects can be made strong and negative by increasing the viscosity of the medium with a combination of cryoprotective mixtures and low temperatures. The use of mixtures with properties very close to water is very promising for conformational studies of enkephalins and of other small linear peptides.     

An opioid growth factor regulates the replication of microorganisms.

An opioid growth factor (OGF), [Met5]-enkephalin, interacts with the zeta (zeta) opioid receptor to modulate development of eukaryotes. We have found that [Met5]-enkephalin, an endogenous opioid peptide serves to inhibit the growth of S. aureus. This effect on growth involves cell proliferative events and is under tonic control, since potent opioid antagonists accelerate cell replication. Both the OGF and zeta opioid receptor were associated with these microorganisms. Other opioid receptors (mu, delta and kappa) were not detected. OGF also controlled the growth of other bacteria: P. aeruginosa and S. marcesans. These results indicate that OGF and its receptor, known to be important in the regulation of mammalian development, also function in the growth of simple unicellular organisms. We suggest that the endogenous opioid system related to growth originated billions of years ago.     

Evidence that endogenous 6-(ARG or LYS)-opioid peptides can interact with kappa-receptors as agonists

Evidence is provided for the abilities of endogenous 6-(Arg or Lys)-opioid peptides to interact with kappa-receptors as agonists. Dynorphin-(1-17) and -(1-8), alpha- and beta-neo-endorphin, [Met5]-enkephalin-Arg6-Phe7 and des acetyl salmon endorphin I significantly inhibited the electrically-evoked contractions of rabbit vas deferens which had been shown to contain kappa-receptors exclusively, indicating that endogenous 6-(Arg or Lys)-opioid peptides could act on kappa-receptors as agonists. Additionally, the inhibition of contractions of rabbit vas deferens by 6-(Arg or Lys)-opioid peptides was antagonized more effectively by Mr 2266 which had a high affinity to both mu- and kappa-receptors, than naloxone which had a high affinity only to mu-receptors. This also suggested that 6-(Arg or Lys)-opioid peptides acted as kappa-receptor agonists. The rank order of the inhibitory potency of 6-(Arg or Lys)-opioid peptides against contractions of rabbit vas deferens was as follows: dynorphin-(1-17) greater than alpha-neo-endorphin greater than beta-neo-endorphin .=. dynorphin-(1-8) greater than des acetyl salmon endorphin I greater than [Met5]-enkephalin-Arg6-Phe7. Since other endogenous opioid peptides such as [Met5]- and [Leu5]-enkephalin and beta-endorphin have been shown not to act on kappa-receptors as agonist, data in the present study suggest that endogenous opioid peptides can be classified into two groups in terms of an ability to interact with kappa-receptors as an agonist.     

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5- and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.     

Binding characteristics of dermorphin, and [dermorphin1-7]-beta c-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe4-Met5]-enkephalin. Synthesis, purification by high performance liquid chromatography and characterization

A photoreactive [D-Ala2, p-N3-Phe4-Met5]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C18 muBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala2, p-N3-Phe4-Met5]enkephalin was 4-fold more potent than [D-Ala2, Met5]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.     

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.     

Mu1: a very high affinity subtype of enkephalin binding sites in rat brain

Displacement studies of [3H]-[D-Ala2-MePhe4-Gly-ol5]-enkephalin ([3H]-DAGO) and [3H]-[D-Ala2-D-Leu5]-enkephalin ([3H]-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the mu, delta and mu 1 sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively.     

The in vitro and in vivo resistance of synthetic enkephalin analogs to three enkephalin-hydrolyzing enzymes

The magnitude of the in vitro and in vivo resistance of 3 synthetic enkephalin analogs, [D-Ala2,Met5]-enkephalin (DAME), [D-Ala2,Met5]-enkephalinamide (DAME-NH2) and [D-Ala2,D-Met5]enkephalin (DADME), to 3 enkephalin-hydrolyzing enzymes, amastatin-sensitive aminopeptidase (AsA), phosphoramidon-sensitive endopeptidase-24.11 (PsE) and captopril-sensitive dipeptidyl carboxypeptidase I (CsD), was estimated by comparing the potency of enkephalins in the absence of the peptidase inhibitor (PI) with that in the presence of the PI. The enhancement of the potency of enkephalins in the isolated mouse vas deferens preparation by 3 PIs, amastatin, phosphoramidon, and captopril, indicated that the resistance of enkephalins to AsA, PsE, or CsD was DADME [symbol: see text] DAME-NH2 [symbol: see text] DAME > [Met5]-enkephalin (ME), DADME > DAME-NH2 > DAME [symbol: see text] ME, or DADME [symbol: see text] DAME-NH2 > DAME > ME, respectively. Additionally, the data obtained by the s.c. administration of enkephalin analogs to 10-day-old rats with or without PI, showed that PsE played the most important role in the inactivation of both DAME and DAME-NH2. In addition to PsE, both AsA and CsD, or AsA alone, played the significant role in the inactivation of DAME, or DAME-NH2, respectively. In the inactivation of DADME, AsA alone played the significant role. These results showed that the 3 peptidases all played important roles in the inactivation of enkephalins, and therefore only an analog like DADME, which was quite resistant to the 3 enzymes, was able to produce the effect without PIs after its systemic administration. Since even DADME was not completely resistant to the 3 enzymes; however, its potency was further increased by pretreatment of infant rats with the PIs.