N-terminal Tetrapeptide Research Articles

Quail (Coturnix japonica) protamine, full-length cDNA sequence, and the function and evolution of vertebrate protamines

Using the chicken protamine gene as a probe, we have isolated and sequenced several positive clones from a quail testis cDNA library which reveal the complete sequence for the quail protamine cDNA. The predicted amino acid sequence for the quail protamine contains the N-terminal tetrapeptide ARYR present in the N-terminal region of the mammalian protamines as well as several conserved motifs and arginine clusters. In addition the size of the quail protamine (56 amino acids) is closer to that of mammals (50 amino acids) than that of the chicken (61 amino acids). Altogether this data strongly suggests the existence of an avian-mammalian protamine gene line during evolution. Southern blot analysis suggests a small number of copies (2) per haploid genome (similar to that of chicken). The reported quail protamine cDNA sequence is the second avian protamine for which the amino acid sequence is available so far and provides new insights into vertebrate protamine function and evolution.

Human gastric lipase. The N-terminal tetrapeptide is essential for lipid binding and lipase activity.

Human gastric lipase subjected to limited tryptic proteolysis lost its ability to hydrolyze emulsified long-chain triacylglycerol. Activity against a water-soluble substrate was however retained, indicating that proteolysis did not affect the active site. Sequence analysis revealed that trypsin specifically cleaved the linkage between lysine-4 and leucine-5. This cleavage rendered the enzyme unable to bind to emulsified triacylglycerol particles, e.g. human milk fat globules. We suggest that the N-terminal tetrapeptide, in particular lysine-4, is essential for the binding of human gastric lipase to lipid/water interfaces, and hence, for its physiological function.

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores.

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.

Monoclonal antibodies to dalargin--a synthetic analog of enkephalins.

Monoclonal antibodies (MABs) to a new drug Dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) enhancing ulcer healing have been produced. Dalargin is a synthetic analog of Leu-enkephalin. With about 40 compounds tested in competition radioimmunoassay it has been shown that specificity of the MABs is directed against the N-terminal tetrapeptide of the molecule. The MABs are sensitive to amino acid substitutions in any of the positions of the fragment and have no cross-reactivity with endogenous opioids. Their further application in pharmacokinetic studies in humans and for characterization of opioid receptors is discussed.

Myelin basic protein binds GTP at a single site in the N-terminus

Myelin basic protein from normal human brain was ADP-ribosylated with Cholera toxin, but not with Pertussis toxin. It bound azido-GTP at a single site in the N-terminal tetrapeptide at the Gln residue. The binding was considerably reduced when GppNHp was present during azido-GTP binding and totally inhibited when GTP gamma S was present. The relevance of this specific binding is not understood at this time.

ChemInform Abstract: Synthesis and Biological Activity of Partially Modified Retro‐Inverso Pseudopeptide Derivatives of the C‐Terminal Tetrapeptide of Gastrin.

ChemInformVolume 18, Issue 47 Natural Products ChemInform Abstract: Synthesis and Biological Activity of Partially Modified Retro-Inverso Pseudopeptide Derivatives of the C-Terminal Tetrapeptide of Gastrin. M. RODRIGUEZ, M. RODRIGUEZ Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorP. DUBREUIL, P. DUBREUIL Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorJ.-P. BALI, J.-P. BALI Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorJ. MARTINEZ, J. MARTINEZ Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this author M. RODRIGUEZ, M. RODRIGUEZ Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorP. DUBREUIL, P. DUBREUIL Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorJ.-P. BALI, J.-P. BALI Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this authorJ. MARTINEZ, J. MARTINEZ Dep. Ferment. Technol., Fac. Eng., Hiroshima Univ., Higashihiroshima 724Search for more papers by this author First published: November 24, 1987 https://doi.org/10.1002/chin.198747319Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue47November 24, 1987 RelatedInformation

Peripheral opioid receptors mediate gastrointestinal secretory and motor effects of dermorphin N-terminal tetrapeptide (NTT) in the dog

Dermorphin N-terminal-tetrapeptide-amide (NTT) increased both basal and pentagastrin- or histamine-induced secretion in conscious dogs chronically implanted with both gastric fistulae and heidenhain pouches. These excitatory effects were significantly prevented by the opioid receptor antagonists naloxone and N-methyl-levallorphan-methanesulphonate. In conscious dogs fitted with electrodes and strain-gauges in different parts of gastrointestinal tract, a premature phase III of the migrating myoelectric complex (MMC) in the duodeno-jejunum was triggered by NTT, while the activity of the antrum was not significantly modified. Further, the peptide enhanced the contractile activity of both proximal and distal portions of the colon, including a long-lasting period of increased muscle tone on the distal colon. Either naloxone or N-methyl-levallorphan-methanesulphonate completely prevented motor effects of NTT on gastrointestinal tract. It is concluded that NTT displays significant opiate-like activity on gastric acid secretion and intestinal motility of the dog by activating peripheral μ opioid receptors.

Structure-activity relationship of dermorphin on gastric secretion.

The amphibian skin heptapeptide dermorphin (DM) administered intracerebroventricularly to rats significantly reduces gastric secretion. Dermorphin and 19 DM homologs and analogs were tested for their effect on gastric volume, pH, H+ ion concentration, and gastric acid output. DM, DM N-terminal pentapeptide and tetrapeptide amides, [D-Met2]DM, [Sar4]DM, [Trp5]DM, [Phe5]DM, [Gly7]DM, [Ser(Bzl)7]DM, and deamidated-DM significantly (P less than 0.01) reduced gastric acid output 2 h after injection. These data provide evidence for the following conclusions on the effect of DM on gastric secretion: ability to inhibit gastric secretion depends on the presence of the D-isomer of Ala at position 2, since [L-Ala2]DM is inactive; the shortest sequence with significant bioactivity is DM N-terminal tetrapeptide amide; the single replacement of amino acid residues in DM elicits a wide range of activities, varying from full biological activity of [Gly7]DM to those analogs with a complete lack of activity, such as [Pro4]DM and [Gly6]DM; and 4) coupling of protective groups to amino and hydroxyl groups of DM results in a significant loss of activity.

I. Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores

AspThrMetArg CysMetValGlyArgValTyrArgProCys TrpGluVal (melanin concentrating hormone, MCH) and several fragment analogs (MCH 1−14, MCH 5−17, MCH 5−14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH 1−17 (10 −12M) >< MCH 5−17 (10 −12M) > MCH 1−14 (10 −11M) > MCH 5−14 (2 × 10 −10M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of ≺-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH 1−14 fragment analog. The MCH activities of the MCH 5−17 and MCH 5−14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self- antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.

Synthesis and biological activity of partially modified retro-inverso pseudopeptide derivatives of the C-terminal tetrapeptide of gastrin.

The effects of partial retro-inverso modifications of selected peptide bonds of the N-terminal tetrapeptide of gastrin have been studied. In some of the synthesized compounds, the phenylalanyl residue has been replaced by the (R,S)-2-benzylmalonyl, 3-phenylpropionyl, benzylcarbamoyl, or benzyloxycarbonyl moieties. All pseudopeptides showed affinity for the gastrin receptor, in vitro, with potencies varying from IC50 = 10(-7) to IC50 = 10(-4) M. These compounds exhibited little or no activity on acid secretion in the anesthetized rat but were able to antagonize the action of gastrin. Among the most potent were Boc-Trp-Leu-gAsp-CO-CH2CH2C6H5 (20) (ED50 = 0.15 microM/kg), Boc-Trp-Leu-gAsp-m(R,S)Phe-NH2 (3) (ED50 = 0.15 microM/kg), and Boc-Trp-gLeu-D-Asp-m(R,S)Phe-NH2 (7) (ED50 = 0.3 microM/kg).

An NMR Study of the Conformations of N-terminal Substance P Fragments and Antagonists

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different 1D and 2D NMR techniques. Total nonexchangeable 1H-NMR assignments were carried out in D2O and the NH protons were assigned in H2O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP1-4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.

Potentiation of dermorphin-induced antinociception by peptidase inhibitors.

We have investigated the effect of amastatin, an aminopeptidase inhibitor, and captopril, an angiotensin converting enzyme inhibitor, on the antinociceptive activity induced by intracerebroventricular administration of dermorphin, a heptapeptide. In addition, the potency of dermorphin was compared with that of one of its metabolites, N-terminal tetrapeptide (Tyr-D-Ala-Phe-Gly), by using the tail pressure test. The antinociceptive activity induced by dermorphin was not potentiated by simultaneous administration of amastatin or captopril. However, there was potentiation when dermorphin was combined with both peptidase inhibitors. Moreover, the N-terminal tetrapeptide was 84 times less potent than its parent heptapeptide when administered intracerebroventricularly. The results suggest that the cleavage of Tyr1-D-Ala2 and Gly4-Tyr5 bonds by brain endopeptidase modulates dermorphin-induced antinociceptive activity.

Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin

A high-performance liquid chromatographic (HPLC) method for the separation of the opioid heptapeptide dermorphin and related fragments has been developed. The chromatographic system was applied in the study of the kinetics of degradation of dermorphin (Der) in various tissues. Der was found to be extremely resistant to human and rat plasma ( T 1 2 >180 min ). Upon incubation with homogenates of rat brains and kidneys, Der was cleaved with a half-life of 20.8±2.2 min and 2.4±0.3 min respectively. The catabolite formed was identified, in both tissues, as the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH. The stability to rat kidney and brain of the N-terminal hexa- and pentapeptides and of the [ 4ψ 5 , NHCO] Der analogue was also investigated. The nature of the enzyme systems involved in the in vitro degradations is discussed.

The site of cleavage in human alpha chains of IgA1 and IgA2:A2m(1) allotype paraproteins by the clostridial IgA protease

Fc fragments of human immunoglobulin A(IgA) of IgAl subclass and IgA2 subclass of A2m(1) allotype were prepared from IgA paraproteins by digestion with a protease from Clostridium sp. (M.O.-6). The N-terminal tetrapeptide of Val-Pro-Ser-Thr- for the Fc of IgA1 subclass, and that of Val-Pro-Pro-Pro- for the Fc of IgA2:A2m(1) allotype, were identified by sequence analysis. The site of cleavage by the protease was defined to be at the Pro-Val peptide bond, which is a common peptide bond present at 221–222 in both alpha chains. IgA of IgA2 subclass of A2m(2) allotype is resistent to the protease due to the different, Arg-Val, peptide bond at the same position.

ACTH 1–4 potentiates α-MSH-induced melanophore dispersion and excessive grooming

The biological activity and a possible modulatory role of the N-terminal tetrapeptide Ser-Tyr-Ser-Met from α-MSH/ACTH was tested in the Anolis melanophore assay, the Xenopus melanophore assay, tyrosinase stimulation in mouse melanoma cells and in excessive grooming in the rat. ACTH 1–4 did not exhibit biological activity in any of these four assays nor did it have modulatory properties in the Xenopus and the melanoma cell assay. However, in the Anolis assay ACTH 1–4 potentiated pigment dispersion induced by α-MSH, α-MSH 5–13 and ACTH 1–24 by a factor of about 2. In the grooming assay ACTH 1–4 potentiated the effects of α-MSH, α-MSH 5–13, ACTH 1–16 and ACTH 5–16, but not those of ACTH 1–24. Oxidized ACTH 1–4 was without biological activity and potentiating properties in all four assays. This study shows that small fragments of the pro-opiomelanocortin precursor, which are devoid of biological activity, can modulate peripheral and central actions of α-MSH/ACTH.

ChemInform Abstract: SYNTHESIS AND PHARMACOLOGICAL ACTIVITY OF PARTIALLY MODIFIED RETRO‐INVERSO DERMORPHIN TETRAPEPTIDES

AbstractEntsprechend dem Formelschema werden die im Titel genannten Tetrapeptide, z.B. (IVb), (Vb) und (VII), hergestellt.

On the degradation of dermorphin and D-Arg 2-dermorphin analogs by a soluble rat brain extract

Degradation of dermorphin, [D-Arg 2]dermorphin and [D-Arg 2, Gly 3, Phe 4]dermorphin in a soluble rat brain extract was examined. The former two heptapeptides were degraded in a similar fashion to produce corresponding N-terminal tetrapeptide as the main degradation product along with the parallel release of Tyr 5, Pro 6 and Ser 7-NH 2. Tyr-D-Arg-Phe-Gly showed a good enzymatic stability. When captopril, an angiotensin-converting enzyme inhibitor, was present in the incubation mixture, hydrolysis of the Gly 4-Tyr 5 bond was markedly suppressed and resulted in release of the corresponding N-terminal hexapeptide as the main degradation product. Combined use of captopril and amastatin, an aminopeptidase inhibitor, markedly suppressed the hydrolysis of these peptides. On the other hand, [D-Arg 2, Gly 3, Phe 4]dermorphin was hydrolyzed easier than the other two heptapeptides and considerable amounts of Tyr 1 and Phe 4 were released after 20 hr incubation while the N-terminal tetrapeptide, Tyr-D-Arg-Gly-Phe, showed a good enzymatic stability. On the basis of these results, possible degradation pathways of these heptapeptides were discussed.

Synthesis and pharmacological activity of partially modified retro-inverso dermorphin tetrapeptides.

We studied the effect of partial retro-inverso modification of selected peptide bonds of N-terminal tetrapeptide analogues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2). Among the 14 compounds synthesized and tested for opioid activity, some tetrapeptides have the C-terminus carrying different amide moieties; retromodifications concern the Phe-Gly bond (Ia-f) and/or the C-terminal carboxamide function (IIIa-d, IIa-d). All pseudotetrapeptide derivatives showed opioid activity in vitro and in vivo. The most potent compounds (II) have a biological potency comparable with that of the original tetrapeptides in the guinea pig ileum preparation and in the mouse tail-flick test after intracerebral or subcutaneous administration.

The analgesic activity of D-Arg 2-dermorphin and its N-terminal tetrapeptide analogs after subcutaneous administration in mice

D-Arg 2-dermorphin and its nineteen N-terminal tetrapeptide analogs were prepared, and their analgesic activities after subcutaneous administration in mice and the stability of a D-Arg 2-dermorphin tetrapeptide to enzymatic degradation were examined. The analgesic effect was assessed by the tail pressure test. D-Arg 2-dermorphin was found to have analgesic potency equal to or slightly greater than that of dermorphin. In a series of tetrapeptide analogs, a very pronounced activity greater than that of morphine was observed for analogs of the following structure, H-Tyr-D-Arg-Phe-X-OH (X = Gly, sarcosine and D-Ala) or its esters. Replacement of D-Arg 2 by D-Arg(NO 2), D-homoarginine or D-Lys resulted in a decrease in potency, suggesting that the guanidino group and side chain length of D-Arg 2 are of great importance for a higher activity. D-Arg 2-tetrapeptide (H-Tyr-D-Arg-Phe-Gly-OH) was found to be more stable than the parent tetrapeptide (H-Tyr-D-Ala-Phe-Gly-OH) to cleavage both by aminopeptidase M and by carboxypeptidase Y.

Studies on analgesic oligopeptides. III. Synthesis and analgesic activity after subcutaneous administration of [D-Arg2]dermorphin and its N-terminal tetrapeptide analogs.

[D-Arg2] dermorphin and nineteen N-terminal tetrapeptide analogs were synthesized by a conventional solution method and their analgesic activities after subcutaneous administration to mice were examined. The analgesic effect was assessed by means of the tail pressure test. [D-Arg2] dermorphin was found to have analgesic potency equal to or slightly greater than that of dermorphin. The N-terminal tetrapeptide, H-Tyr-D-Arg-Phe-Gly-OH (I), showed a potency 4.8 times that of morphine and comparable with that of dermorphin on a molar basis. Among the tetrapeptide analogs, several analogs in which Gly4 was replaced by sarcosine or D-Ala exhibited very potent activity more than that of I. On the other hand, replacement of Gly4 by Pro, Leu or D-Leu resulted in a marked decrease in potency. Replacement of either Phe3 by other aromatic amino acid or D-Arg2 by other basic D-amino acid gave analogs with greatly decreased activities. However, one analog whose guanidino functionality on D-Arg2 was blocked by a nitro group, showed activity one-third that of the parent peptide (I). On the basis of these results, the structure-activity relationship for the tetrapeptide is discussed.