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.     

Expression of opioid genes in bovine seminal vesicles

In seminal vesicles, the organ producing most of seminal plasma in the bovine species, the pro-opiomelanocortin and the proenkephalin genes are transcribed and translated, and their translation products processed into opioid peptides, which are secreted into the seminal plasma. By using a micro-organ preparation of seminal vesicles we found that, after 20 h of incubation with labelled methionine, a multiplicity of opioids was produced. Among these, [Met]enkephalin and beta-endorphin were positively identified, whereas in the newly formed secretion only [Met]enkephalin was detected. This may be correlated to the finding that the concentration of beta-endorphin in an extract of seminal plasma was one order of magnitude lower than that of [Leu]enkephalin and [Met]enkephalin. These findings expand the picture of the presence of opioid peptides in the male reproductive tract, indicating that they should have a role(s) in the physiology of reproduction, not only in the hypothalamus-pituitary-gonadal axis, determining the reproductive potential, but also in the so-termed sex accessory glands, determining the actual events leading to reproduction. To our knowledge this is also the first case studied of opioid peptides produced as exocrine hormones.     

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.     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Studies on the metabolic interactions between 4-methylpyrazole and methanol using the monkey as an animal model

Bovine adrenal chromaffin granules have been shown to contain [Met]enkephalin and [Leu]enkephalin and at least seven other small peptides that exhibit specific binding to opiate receptors. Six of these peptides have been characterized and their structures established as (O)-[Met]enkephalin, [Met]enkephalin-Arg⁶-Arg⁷, [Met]enkephalin-Lys⁶, [Met]enkephalin-Arg⁶, [Leu]enkephalin-Arg⁶, and [Met]enkephalin-Arg⁶-Phe⁷. Many of these hexa- and heptapeptides are also present in bovine and human brain. It is suggested that the presence of these peptides in a tissue is evidence of a common biosynthetic pathway of the enkephalins from a large precursor protein.     

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.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

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.     

Binding of beta-endorphin and its fragments to the non-opiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid beta-endorphin receptor the decapeptide immunorphin ([3H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [3H]immunorphin binds with a high affinity to the non-opioid beta-endorphin receptor of mouse peritoneal macrophages (Kd 2.4 +/- 0.1 nM). The specific binding of [3H]immunorphin to macrophages was inhibited by unlabeled beta-endorphin (Ki of the [3H]immunorphin-receptor complex 2.9 +/- 0.2 nM) and was not inhibited by unlabeled naloxone, alpha-endorphin, gamma-endorphin, and [Met5]enkephalin (Ki > 10 microM). Thirty fragments of beta-endorphin were synthesized, and their ability to inhibit the specific binding of [3H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as beta-endorphin is its fragment 12-19 (Ki 3.1 +/- 0.3 nM).     

Enkephalin hydrolysis by mouse plasma in vitro.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyr-containing metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatin-sensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma.     

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.     

Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.     

Interaction of iodinated human [D-Ala2]beta-endorphin with opitate receptors.

The interaction of beta-endorphin with opiate receptors was studied by using the radioiodinated, metabolically stable D-Ala2 derivative of human beta-endorphin. This analog binds specifically to rat brain membrane preparations with an apparent Kd of about 2.5 x 10-9 M. The ability of various enkephalin analogs, as well as opiate agonists and antagonists, to inhibit the binding of beta-endorphin clearly demonstrates that this peptide can bind to opiate receptors. However, the effects of various cations on the binding of 125I-[D-Ala2]beta-endorphin are markedly different from those found for enkephalin binding. Sodium ion at physiological concentrations decreases substantially the binding of enkephalins but only slightly decreases endorphin binding, whereas manganese enhances enkephalin binding but has no effect on endorphin binding. Moreover, potassium (100 mM) decreases the binding of beta-endorphin but does not affect enkephalin binding. These results suggest that beta-endorphin and enkephalin bind differently to the same receptor or bind to different receptors with overlapping specificity.     

Characterization and immunocytochemical application of monoclonal antibodies against enkephalins

Monoclonal antibodies were produced following immunization of mice with either [Leu5]enkephalin-bovine serum albumin or [Met5]enkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu- and Met-enkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did not recognize other endogenous peptides. In immunocytochemistry the NOC1 and NOC2 recognized all well-established "enkephalin immunoreactive sites," but they did not bind to areas known to contain beta-endorphin or high levels of pro-enkephalin. NOC1 was shown to be a suitable tool to demonstrate enkephalin immunoreactive sites by radioimmunocytochemistry utilizing both internally and externally labeled monoclonal antibodies.     

Characteristics of Non-opioid β-Endorphin Receptor

Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]pentarphine (VKPFY) (Ki values were 0.0060 +/- 0.0004, 2.7 +/- 0.2, 2.6 +/- 0.2, and 2.8 +/- 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 +/- 44.7, 698.6 +/- 28.1, 279.1 +/- 15.4, and 172.2 +/- 1.8 fmol/mg protein, respectively. Unlabeled beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.     

Electrically-Induced Release of Opioid Peptides from the Guinea-Pig Myenteric Plexus Preparation

Abstract

Preparations of guinea-pig myenteric plexus-longitudinal muscle suspended in Krebs solution were stimulated electrically in the presence of cycloheximide and tetraethylammonium. The amounts of eleven endogenous opioid peptides released into the perifusing Krebs solution were determined and correlated with the decrease in the tissue contents induced by stimulation. For pro-enkephalin fragments the ratio of release to reduction in tissue contents was 29 to 43% for [Met]enkephalin, [Leu]enkephalin, [Met]enkephalyl-RF and [Met]enkephalyl-RGL. With [Met]enkephalyl-RRV-NH₂ (BAM-8) the ratio was higher by 50% or more. However, it is of interest that there was no release of the probable precursor [Met]enkephalyl-RRVGRPEWWMDYQ(BAM-18). In this context it may be important that BAM-8 is the only endogenous opioid peptide having-NH₂ at the C-terminal. The low tissue levels of pro-dynorphin derived peptide have made estimation of release unreliable.     

Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties, and regional distribution in human brain

Abstract: The compound [³H-Tyr¹,D-Ala²,Lcu-OH⁵]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase ( enkephalinase ) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [³H]Tyr-D-Ala-Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly³-Phe⁴ amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5-enkephalin levels.     

Secondary structure determination of human beta-endorphin by 1H NMR spectroscopy

The 1H NMR spectra of human beta-endorphin indicate that the peptide exists in random-coil form in aqueous solution but becomes helical in mixed solvent. Thermal denaturation NMR experiments show that in water there is no transition between 24 and 75 degrees C, while a slow noncooperative thermal unfolding is observed in a 60% methanol-40% water mixed solvent in the same temperature range. These findings are consistent with circular dichroism studies by other workers concluding that beta-endorphin is a random coil in water but that it forms 50% alpha-helix or more in mixed solvents. The peptide in the mixed water-methanol solvent was further studied by correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments. These allow a complete set of assignments to be made and establish two distinct stretches over which the solvent induces formation of alpha-helices: the first occurs between Tyr-1 and Thr-12 and the second between Leu-14 and extending to Lys-28. There is evidence that the latter is capped by a turn occurring between Lys-28 and Glu-31. These helices form at the enkephalin receptor binding site, which is at the amino terminus, and at the morphine receptor binding site, located at the carboxyl terminus [Li, C. H. (1982) Cell (Cambridge, Mass.) 31, 504-505]. Our findings suggest that these two receptors may specifically recognize alpha-helices.