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PubMedDate not supplied

A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function.

Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to inhibit gonadotropin release from the cultured anterior pituitary. This peptide is the first hypothalamic peptide that inhibited gonadotropin release reported in vertebrates. We, therefore, termed it gonadotropin-inhibitory hormone (GnIH). After this finding, we found that GnIH-related peptides were present in the brains of other vertebrates, such as mammals, amphibians, and fish. These GnIH-related peptides possessed a LPXRF-amide (X=L or Q) motif at their C-termini in all investigated animals. Mass spectrometric analyses combined with immunoaffinity chromatography were powerful techniques for the identification of mature endogenous LPXRF-amide peptides. The identified LPXRF-amide peptides were found to be localized in the hypothalamus and brainstem areas, and to regulate pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and function of a new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides in vertebrates. Recent studies on the receptors for LPXRF-amide peptides will also be reviewed.

Amides↗
PubMed1998

Identification of glycine-extended CCK peptides in endocrine cells and modulation of CCK amide and CCK Gly content and secretion from endocrine tumor cells by an inhibitor of amidation.

Immunoreactive glycine-extended CCK peptides are found in normal mouse cerebral cortex and are very abundant in some CCK expressing endocrine tumor cells in culture. The glycine-extended forms in mouse cortex and in cell lines mirror their respective amidated forms. Mouse cerebral cortex, mouse AtT20 and rat WE cells produce mainly CCK 8 amide and CCK 8 Gly. In contrast, mouse intestinal STC-1 cells produce CCK 22 and CCK 8 amide along with forms of CCK Gly which are slightly larger than their respective amidated forms. The CCK 8 Gly-like peptide from AtT20 cells, after desulfation, co-eluted on HPLC with unsulfated CCK 8 Gly. Addition of copper and ascorbate to culture medium of WE cells caused a small increase in secretion of amidated CCK, without changing cellular levels of this peptide. Treatment with the amidation inhibitor diethyldithiocarbamate greatly decreased cellular content and secretion of CCK amide while it increased cellular content and secretion of CCK Gly. These results provide further evidence that glycine-extended CCK peptides are the immediate precursors of amidated CCK peptides.

Amides↗
PubMed1995

Peptide models of helical hydrophobic transmembrane segments of membrane proteins. 1. Studies of the conformation, intrabilayer orientation, and amide hydrogen exchangeability of Ac-K2-(LA)12-K2-amide.

The secondary structure, amide hydrogen exchangeability, and intramembrane orientation of the hydrophobic peptide Ac-K2-(LA)12-K2-amide [(LA)12] were studied by a combination of circular dichroism (CD), Fourier transform infrared (FTIR), and proton nuclear magnetic resonance (1H NMR) spectroscopic techniques. All three techniques indicate that (LA)12 adopts predominantly helical conformations in various organic solvents, detergent micelles, and phospholipid bilayers. Also, attenuated total reflectance FTIR studies of oriented phospholipid bilayers demonstrate that (LA)12 is arranged with the long helical axis perpendicular to the bilayer plane. FTIR and 1H NMR studies of the exchangeability of the amide protons of (LA)12 indicate that in all media there are at least two populations of amide protons which exchange with the bulk solvent at markedly different rates. Moreover, the 1H NMR spectroscopic studies indicate that, in organic solvents and micellar dispersions, amide proton exchange rates decrease progressively from the N- or C-terminus of the peptide toward the central region. Our results are thus consistent with (LA)12 retaining a predominantly helical structure with so-called frayed ends in all media. The amide proton exchange studies also indicate that when (LA)12 is dispersed in lipid bilayers, the slowly exchanging population of amide protons is larger than that observed in organic solvents or in micellar dispersions and that most of that proton population is virtually unexchangeable. Such observations are consistent with the sequestration of the central regions of the peptide in the hydrophobic domains of the lipid bilayer. The CD and FTIR data indicate that although (LA)12 seems to retain conformations with a high alpha-helical content in all media examined, its conformation is sensitive to the composition of the surrounding medium, in contrast to the polyleucine-based analogues which have been studied previously. In particular, the FTIR spectroscopic data indicate that (LA)12 may exhibit an amide I absorption band between 1633 and 1639 cm-1 under some circumstances. The relative intensity of this band changes with the composition of the surrounding medium and its appearance has previously been correlated with the formation of 3(10)-helical structures [Miick et al. (1992) Nature 359, 653-655]. Thus (LA)12 may be interconverting between different helical conformations in response to changes in the physical properties of the medium in which the peptide is dispersed. Our results suggest that (LA)12 should serve as a good peptide model of hydrophobic, transmembrane helices which are conformationally sensitive to the properties of the lipid bilayer in which they reside.

Alanine↗
PubMed1989

Retinobenzoic acids. 4. Conformation of aromatic amides with retinoidal activity. Importance of trans-amide structure for the activity.

N-Methylation of two retinoidal amide compounds, 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]benz oic acid (3, Am80) and 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carbonyl]amino]benzoic acid (5, Am580), resulted in the disappearance of their potent differentiation-inducing activity on human promyelocytic leukemia cell line HL-60. Studies with 1H NMR and UV spectroscopy indicated that large conformational differences exist between the active secondary amides and the inactive N-methyl amides. From a comparison of the spectroscopic results of these amides with those of stilbene derivatives, the conformations of the active amides are expected to resemble that of (E)-stilbene, whereas the inactive amides resemble the Z isomer: 3 (Am80) and 5 (Am580) have a trans-amide bond and their whole structures are elongated, while the N-methylated compounds [4 (Am90) and 6 (Am590)] have a cis-amide bond, resulting in the folding of the two benzene rings. These structures in the crystals were related to those in solution by 13C NMR spectroscopic comparison between the two phases (solid and solution).

Amides↗
PubMed2001

Characterization of glutathione amide reductase from Chromatium gracile. Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling.

Among the Chromatiaceae, the glutathione derivative gamma-l-glutamyl-l-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 microm for glutathione amide disulfide and NADH, respectively (in contrast to K(m) values of 6.9 mm for glutathione disulfide and 1.98 mm for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.

Amino Acid Sequence↗
PubMed1996

Fatty acid amide biosynthesis: a possible new role for peptidylglycine alpha-amidating enzyme and acyl-coenzyme A: glycine N-acyltransferase.

Fatty acid primary amides have recently been recognized as mammalian hormones [Cravatt et al. (1995) Science 268, 1506-1509]. The route to their biosynthesis is unknown. Many mammalian peptide hormones also possess a C-terminal alpha-amide moiety that arises from the posttranslational oxidative cleavage of a C-terminal glycine-extended precursor. The enzyme that catalyzes this reaction is peptidylglycine alpha-amidating enzyme, which is known to preferentially amidate peptide substrates containing a penultimate, hydrophobic amino acid [Tamburini et al. (1990) Int. J. Pept. Protein Res. 35, 153-156]. We show that N-myristoylglycine is a substrate for peptidylglycine alpha-amidating enzyme with a (V/K)app that is 55 +/- 4% of the value measured for D-Tyr-Val-Gly. N-Fatty acylglycines are enzymatically produced in mammals from fatty acyl-coenzyme A (CoAs) and glycine by acyl-CoA:glycine N-acyltransferase. The sequential actions of acyl-CoA:glycine N-acyltransferase and peptidyl-glycine alpha-amidating enzyme would lead to the biosynthesis of fatty acid amides.

Acyltransferases↗
PubMed1993

Determination of the rate constants k1 and k2 of the Linderström-Lang model for protein amide hydrogen exchange. A study of the individual amides in hen egg-white lysozyme.

The pH dependence of the amide/solvent hydrogen exchange of individual amide groups in hen egg-white lysozyme has been studied by nuclear magnetic resonance spectroscopy. Lysozyme has been used here as a model for a globular protein to re-examine the hypothesis for the amide/solvent hydrogen exchange reaction proposed by K. Linderstrøm-Lang and described in detail by Hvidt and Nielsen. The work has been focused on the most slowly exchanging amide at the temperature of 21 degrees C and in the pH range between 4 and 8. Exchange rates have been measured for 64 of the 126 amide protons and the pH dependence has been determined for 52 of these. The amides examined represent a sample that includes all the types of secondary structure and they are placed in the globular structure in a range of 3.2 A to 8.5 A from the closest water molecule on the surface. The measured exchange rates at pH 6 have been compared to these structural parameters and the results suggest that the rate constants are determined partly by the distance to the surface and partly by the type of secondary structure the amide is engaged in. Near the surface and in the very interior the distance to the surface seems to be rate-determining. Between the extremes the type of secondary structure is rate determining. The pH dependent exchange of the examined amides was shown to be in agreement with the Linderstrøm-Lang model. For each of the amides examined the rate constants for the opening and the closing reaction in the first reaction step of the Linderstrøm-Lang model has been calculated and compared to structural parameters.

Amides↗
PubMed2005

Water-promoted hydrolysis of a highly twisted amide: rate acceleration caused by the twist of the amide bond.

The water-promoted hydrolysis of a highly twisted amide is studied using density functional theory in conjunction with a continuum dielectric method to introduce bulk solvent effects. The aim of these studies is to reveal how the twisting of the C-N bond affects the neutral hydrolysis of amides. To do so, both concerted and stepwise mechanisms are studied and the results compared to the ones from the hydrolysis of an undistorted amide used as reference. In addition, an extra explicit water molecule that assists in the required proton-transfer processes is taken into account. Our results predict important rate accelerations of the neutral hydrolysis of amides when the C-N bond is highly twisted, the corresponding barrier relaxation depending on the specific reaction pathway and transition state involved. Moreover, our calculations strongly suggest a change in reaction mechanism with degree of amide bond twist, and clearly point to a concerted mechanism at neutral pH for the hydrolysis of highly twisted amides. In addition, the twisting of the amide bond also provokes a higher dependence on an auxiliary water molecule for the concerted mechanism, due to the orthogonality of the lone pair of the nitrogen and the carbonyl pi orbital. There is a direct implication of these findings for biological catalytic mechanism of peptide cleavage reactions that undergoes ground-state destabilization of the peptide.

Amides↗
PubMed1987

Comparison of the effects of FMRF-amide and pQDPFLRF-amide on identified Helix neurons.

Intracellular recordings were made from a selection of identified neurons in the sub-oesophageal ganglia of Helix aspersa, and their responses to the molluscan neuropeptides FMRF-amide and pQDPFLRF-amide compared. While FMRF-amide excited certain neurons and inhibited others, pQDPFLRF-amide did not produce excitation in any of the cells tested. A few cells were unresponsive to pQDPFLRF-amide, but most were inhibited with varying potency. FMRF-amide was generally 10-100 times more potent than pQDPFLRF-amide, but one cell, E13 in the visceral ganglion, showed equal sensitivity to the two peptides. On most cells there was no clear evidence for cross desensitization between the two peptides, although it is possible that in some cases where both peptides are inhibitory they may be acting on a single receptor.

Action Potentials↗
PubMed1996

Glucagon-like peptide-1-(9-36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor.

This study assesses the importance of metabolites formed following exogenous administration of glucagon-like peptide-1-(7-36) amide (GLP-1). After subcutaneous (s.c.) administration of GLP-1 to dogs the plasma immunoreactivity of GLP-1 measured by two different radioimmunoassays (RIAs) were higher than that measured by a sandwich enzyme-linked immunosorbent assay (ELISA). This discrepancy was due to the formation of the metabolites GLP-1-(9-36) amide, GLP-1-(7-35) and GLP-1-(7-34). Receptor binding studies using baby hamster kidney cells expressing the human pancreatic GLP-1 receptor showed that the affinity of GLP-1-(9-36) amide, GLP-1-(7-35) and GLP-1-(7-34) was 0.95%, 12% and 2.8%, respectively, of the affinity of GLP-1-(7-36) amide. Furthermore, GLP-1-(9-36) amide was shown to be an antagonist to adenylyl cyclase activity, whereas GLP-1-(7-35) and GLP-1-(7-34) were shown to be agonists. GLP-1-(9-36) amide was shown to be present in vivo in amounts up to 10-fold that of GLP-1-(7-36) amide. Due to its low binding affinity, this antagonistic metabolite does not seem to be able to cause physiological antagonism upon s.c. administration of the peptide.

Animals↗
PubMed1990

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation.

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.

Alkylation↗
PubMed1991

GLP-1-(7-36) amide, -(1-37), and -(1-36) amide: potent cAMP-dependent stimuli of rat parietal cell function.

We investigated the effect of glucagon-like peptide 1 (GLP-1)-(7-36) amide and its molecular variants GLP-1-(1-37) and GLP-1-(1-36) amide on enzymatically dispersed enriched rat parietal cells using [14C]aminopyrine accumulation as a measure of H+ production. GLP-1-(7-36) amide was 100 times more potent than GLP-1-(1-37) and GLP-1-(1-36) amide in stimulating [14C]aminopyrine accumulation. At their maximally effective concentrations, GLP-1-(7-36) amide (10(-8) M), GLP-1-(1-37) (10(-6) M), and GLP-1-(1-36) amide (10(-6) M) reached 80-90% of the response to 10(-4) M histamine. However, the peptides were 100-10,000 times more potent than histamine, which induced maximal [14C]aminopyrine accumulation at 10(-4) M. Stimulation by GLP-1 was dependent on the presence of a phosphodiesterase inhibitor and was not altered by pertussis toxin. Ranitidine failed to affect the response to the GLP-1 variants. Stimulation of H+ production by GLP-1 was accompanied by an increase in the formation of adenosine 3',5'-cyclic monophosphate (cAMP) but not by changes in phosphoinositol breakdown. In stimulating [14C]aminopyrine accumulation, the GLP-1 variants acted additively to threshold but not to maximal concentrations of histamine, suggesting that histamine and GLP-1 activate the same cAMP pool. In contrast, in anesthetized rats GLP-1-(7-36) amide (10-500 ng.kg-1.h-1) had no effect on basal and pentagastrin-stimulated acid secretion in vivo. We conclude that GLP-1 exerts a direct stimulatory effect on rat parietal cells. This potent effect is mediated by cAMP and is independent of H2 receptors. In vivo direct stimulation by GLP-1 of the parietal cells might be counterbalanced by indirect inhibitory mechanisms that are excluded in the in vitro cell system.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Fatty acid amide hydrolase competitively degrades bioactive amides and esters through a nonconventional catalytic mechanism.

The greater reactivity of esters relative to amides has typically been reflected in their faster rates of both solvolysis and enzymatic hydrolysis. In contrast to this general principle, the serine hydrolytic enzyme fatty acid amide hydrolase (FAAH) was found to degrade amides and esters with equivalent catalytic efficiencies. Mutation of a single lysine residue (K142) to alanine (K142A) abolished this property, generating a catalytically compromised enzyme that hydrolyzed esters more than 500-fold faster than amides. Conversion of this same lysine residue to glutamic acid (K142E) produced an enzyme that also displayed severely diminished catalytic activity, but one that now maintained FAAH's ability to react with amides and esters at competitive rates. The significant catalytic defects exhibited by both the K142A and K142E mutants, in conjunction with their altered pH-rate profiles, support a role for lysine 142 as a general base involved in the activation of FAAH's serine nucleophile. Moreover, the dramatically different amide versus ester selectivities observed for the K142A and K142E mutants reveal that FAAH's catalytic efficiency and catalytic selectivity depend on distinguishable properties of the same residue, with the former relying on a strong catalytic base and the latter requiring coupled general acid-base catalysis. We hypothesize that FAAH's unusual catalytic properties may empower the enzyme to function effectively as both an amidase and esterase in vivo.

Acylation↗
PubMed2025

Deazapurine Amide-Bond Synthetases: a New Family of Amide-Bond-Forming Enzymes Driving the Diversity of Peptidyl Deazapurine Natural Products.

Amide bond-forming enzymes play a crucial role in generating structural diversity in natural products by assembling them from relatively simple precursors. Two distinct types of standalone amid-bond-forming enzymes are commonly involved in natural product biosynthesis, including ATP-grasp enzymes and amide bond synthetases. Here, we report a new family of amide bond synthetases that catalyze amide bond formation between deazapurine as the sole carboxylic acid substrate and various amine substrates, which we have designated as deazapurine amide bond synthetases (DABS). This evolutionarily related enzyme family plays a central role in diversifying the structures of peptidyl deazapurine natural products. Our gene mining analysis reveals that most DABS-associated biosynthetic gene clusters (BGCs) remain cryptic. Therefore, systematic characterization of these cryptic BGCs holds great potential for discovering novel peptidyl deazapurine natural products with diverse biological activities.

Biological Products↗
PubMed2000

Hydrolysis behavior of prednisolone 21-hemisuccinate/beta-cyclodextrin amide conjugate: involvement of intramolecular catalysis of amide group in drug release.

Prednisolone 21-hemisuccinate/beta-cyclodextrin (beta-CyD) amide conjugate was prepared by binding prednisolone 21-hemisuccinate covalently to the amino group of mono(6-deoxy-6-amino)-beta-CyD through amide linkage. Prednisolone 21-hemisuccinate was intramolecularly transformed to prednisolone 17-hemisuccinate, and the parent drug, prednisolone, was slowly released from the 21-hemisuccinate with a half life of 69 h in pH 7.0 at 37 degrees C; the drug release at 25 degrees C was less than 10% for 48 h. In sharp contrast, the hydrolysis of prednisolone 21-hemisuccinate/beta-CyD amide conjugate was significantly faster (half life of 6.50 min at 25 degrees C) and gave prednisolone and mono(6-deoxy-6-succimino)-beta-CyD as products. The hydrolysis of the beta-CyD amide conjugate was subject to a specific-base catalysis in the alkaline region. The rapid hydrolysis of the conjugate can be ascribed to the involvement of an intramolecular nucleophilic catalysis of the amide group in the reaction. The succinic acid, bound to a drug through ester linkage at one carboxylic group and bound to a pro-moiety through amide linkage at another carboxylic group, may be useful as a spacer for construction of the immediate release type prodrugs of CyDs.

Amides↗
PubMed1996

A novel biologically active eel calcitonin analogue with carboxyl terminal Hse32-amide: carboxyl terminal Pro32-amide in calcitonin is not essential for biological activity.

Eel calcitonin (CT) analogues having C-terminal homoserine (Hse)-amide at position 31, 32, or 33 were synthesized, and in vivo hypocalcemic activity of the analogues were determined. The present study showed that: (i) An eel CT analogue having Hse-amide at position 32 was more active than native eel CT, and the duration of hypocalcemic action of the analogue was equivalent to that of native eel CT. (ii) Either curtailment or elongation of [Hse32-amide]-eel CT peptide chain by one amino acid resulted in a great loss of hypocalcemic activity. The results of the present study indicate that Pro-amide at the C-terminus of CT is not essential for its biological activity in vivo. Replacement of C-terminal Pro32-amide by Hse32-amide in eel CT molecule produced an analogue with a hypocalcemic activity greater than that of native eel CT.

Amino Acid Sequence↗
PubMed1993

Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting beta-cells.

Exendin-4 purified from Heloderma suspectum venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)-amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced 125I-GLP-1, and unlabeled GLP-1 displaced 125I-exendin-4 from the binding site at rat insulinoma-derived RINm5F cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular cAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on cAMP. They each competed with the radio-labeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin secretion in isolated rat islets, and, in mouse insulinoma beta TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin-(9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist.

Animals↗