Endogenous ligands for κ-opiate receptors

The receptor preferences of opioids in the mouse vas deferens was tested by means of tolerance and cross-tolerance studies. The preparations were rendered tolerant in situ by superfusion with the κ-receptor agonist dynorphin and with α-neoendorphin, respectively, and set up in vitro in the presence of the respective peptide to maintain tolerance. The investigations revealed strong κ-agonistic activities both of α-neoendorphin and of dynorphin and its fragments 1–13 and 1–11. As the dynorphin chain shortened, the κ-receptor activity declined and δ-receptor activity became progressively apparent. Interestingly, the octapeptide met-enkephalin[Arg⁶,Gly⁷,Leu⁸], a fragment of the adrenal medulla proenkephalin, also displayed considerable κ-agonistic properties under the experimental conditions employed. Presumably, the decapeptide α-neoendorphin and the octapeptide met-enkephalin[Arg⁶,Gly⁷,Leu⁸] cover in addition to the κ-receptor population in the MVD further opiate receptors, most probably δ-receptors.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme

Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1-6), dynorphin (1-13), alpha-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid D-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1-13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1-6), alpha-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [delta1-10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10(-10) M range.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Pro-enkephalin gene derived peptides in the porcine stomach: cellular distribution and molecular forms

Antisera to the enkephalin variants Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 have been used in immunohistochemistry and radioimmunoassay studies of hog stomach. In the mucosa of the antrum, but not fundus, there was identified a population of immunoreactive endocrine-like cells. When extracts of antral mucosa were fractionated by gel filtration on Sephadex G50 the predominant immunoreactive forms of Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 were found to elute before the standards, and were compatible with N-terminally extended variants. In the muscle layers of both antrum and fundus, immunoreactive nerve fibers were found, these were especially numerous in the myenteric plexus. In extracts of the antral muscle, 50-60% of both Met-enk Arg6Phe7 and Met-enk Arg6Gly7Leu8 immunoreactivity eluted in the position of the standards and the remainder had the properties of N-terminally extended variants. In the fundus muscle the variants accounted for 70-80% of total activity. The results indicate that the proenkephalin gene is expressed in neurones and endocrine cells of the hog stomach. The different patterns of molecular forms found in different regions of the stomach suggest that the precursor is processed by different pathways in different populations of endocrine cells and neurones.     

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.     

Endopeptidase in human cerebrospinal fluid which cleaves proenkephalin B opioid peptides at consecutive basic amino acids

An endopeptidase releasing the common N-terminal hexapeptide, (Leu)-enkephalin-Arg6, from dynorphins A and B, and alpha-neoendorphin was purified from human cerebrospinal fluid. Purification involved ion-exchange chromatography (DEAE-Sepharose CL-6B), hydrophobic interaction chromatography (phenyl-Sepharose CL-4B) and molecular sieving (Sephadex G-100). The enzyme showed molecular heterogeneity. A major fraction had an apparent molecular weight of about 40,000. It had an optimum activity in the pH range of 6-8. The conversion of dynorphin A was not affected by EDTA or iodoacetate but strongly reduced in the presence of phenylmethyl-sulphonyl fluoride, suggesting the enzyme is a serine protease.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Regional Distribution of Methionine-Enkephalin-Arg6-Phe7 in the Rat Brain: Comparative Study with the Distribution of Other Opioid Peptides

The distribution of the opioid peptide methionine-enkephalin-arginine6-phenylalanine7 (M-Enk-Arg6-Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M-Enk-Arg6-Phe7 has been further characterized by high performance liquid chromatography. The levels of M-Enk-Arg6-Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionine-enkephalin (M-Enk), leucine-enkephalin (L-Enk), dynorphin 1-13, and alpha-neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M-Enk-Arg6-Phe7 immunoreactive material (ir), M-Enk ir, and L-Enk ir. The distribution of dynorphin 1-13 ir and alpha-neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c-DNA coding for the prohormones, in which it has been hypothesized that M-Enk-Arg6-Phe7 and M-Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin-related peptides and alpha-neoendorphin arise from a separate precursor, prodynorphin.     

Characterisation of N-terminally extended met-enkephalin Arg6Gly7Leu8 variants in the porcine upper digestive tract

Proenkephalin A-derived peptides are known to occur in the gut, but their precise identity is uncertain. We report here the isolation of N-terminally extended forms of Met-enkephalin Arg6Gly7Leu8 from porcine upper digestive tract monitored by radioimmunoassay. A single major form was identified in pyloric antral muscle and mucosa, but in the duodenum two major forms were detected. Microsequence analysis together with immunological data revealed that the antral mucosal peptide and the most acidic duodenal peptide had identical amino-acid sequences, corresponding to a 5.3 kDa peptide terminating in Met-enkephalin Arg6Gly7Leu8. The data indicate that high-molecular-weight peptides may constitute a major proportion of gut opioid peptide immunoactivity.     

NMR and molecular dynamics studies of tachykinins: conformation of substance P fragment 4-11

The conformation of the C-terminal octapeptide fragment of Substance P (SP4-11, Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with gamma-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.     

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.     

Subcellular Localization, Partial Purification, and Characterization of a Dynorphin Processing Endopeptidase from Bovine Pituitary

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.     

Direct membrane effects of morphine and endorphins on Amoeba proteus.

Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1-8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.     

NMR and Molecular Dynamics Studies of Tachykinins: Conformation of Substance P Fragment 4-11

Abstract

The conformation of the C-terminal octapeptide fragment of Substance P (SP_4-11, Pro-GlnGln-Phe-Phe-Gly-Leu-Met-NH₂) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with y-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.