N-terminal Pyroglutamyl Residue Research Articles

Mass spectrometric characterisation of the major peptides of the male ejaculatory duct, including a glycopeptide with an unusual zwitterionic glycosylation

Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect. Biological significanceSeminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.

Peptidomics of potato protein hydrolysates: implications of post-translational modifications in food peptide structure and behaviour.

Post-translational modifications (PTMs) often occur in proteins and play a regulatory role in protein function. There is an increasing interest in the bioactivity of food protein-derived peptides, but the occurrence of PTMs and their influence on food peptide structure and behaviour remain largely unknown. In this study, the shotgun-based peptidomics strategy was used to identify the occurrence of PTMs in peptides generated from potato protein hydrolysis using digestive proteases. Diverse PTMs were found in the potato peptides, including acetylation of lysine, N-terminal of proteins and peptides, C-terminal amidation, de-amidation of asparagine/glutamine, methylation and trimethylation, methionine oxidation and N-terminal pyro-glutamyl residue formation. The modifications may have been formed naturally or as a result of chemical reactions during isolation and enzymatic processing of the potato proteins. Most of the PTMs were calculated to decrease the isoelectric point and increase molecular hydrophobicity of the peptides, which will influence their bioactivity while also potentially altering their solubility in an aqueous environment. This is the first study to unravel that food-derived peptides can be widely modified by PTMs associated with notable changes in peptide chemical properties. The findings have broader implications on the bioavailability, biomolecular interactions and biological activities of food peptides.

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 °C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 °C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.

Pyrrolidon Carboxypeptidase Activities in the Hypothalamus-pituitary-thyroid and Hypothalamus-pituitary-ovary Axes of Rats with Mammary Gland Cancer Induced by N-methyl Nitrosourea

Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.

Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Onconase (rONC), otherwise known as ranpirnase or P-30 protein, which was initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We have determined the solution NMR structure of a recombinant onconase with Met(-1), Gln1, and Leu23 residues (M-1, Q1, M23L)rONC. The 20 best solution structures had a backbone root mean square deviation of 0.41 +/- 0.09 A with respect to the average structure. The energy-minimized average NMR structure had a backbone root mean square deviation of 0.72 A from the x-ray crystallographic structure of native onconase; however, the orientation of the N-terminal residue in the two structures was very different. Comparison of the 15N HSQC spectrum of (M-1, Q1, M23L)rONC with that of a mutant E1S-rONC, which is identical to the nONC except with the N-terminal pyroglutamyl residue replaced by Ser, showed that N-terminal and residue 23 mutations induced structural changes in regions beyond the mutation sites. Model-free analysis of the backbone amide 15N-T1, 15N-T2, and 15N-1H NOE relaxation data for (M-1, Q1, M23L)rONC and E1S-rONC revealed that the E1S-rONC molecule showed very little flexibility, whereas (M-1, Q1, M23L)rONC exhibited substantial flexibility, which may account for the previously observed reduced stability and increased protease susceptibility. The alpha1 helix and beta-sheets of (M-1, Q1, M23L)rONC displayed bending motions. These data provided strong evidence for the presence of an N-terminal hydrogen bond network in E1S-rONC, but not in (M-1, Q1, M23L)rONC.

Orexins/hypocretins: waking up the scientific world.

Orexins/hypocretins: waking up the scientific world.

Development of a Continuous, Fluorometric Coupled Enzyme Assay for Thyrotropin-Releasing Hormone-Degrading Ectoenzyme

Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4.19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80°C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave Km values for TRHMCA of 3.4 ± 0.7 μM (n = 5) and 3.8 ± 0.5 μM (n = 5), respectively. Ki values determined by the discontinuous assay for TRH and TRH-OH were 35 ± 4 μM (n = 3) and 311 ± 31 μM (n = 5), respectively.

N-terminal modifications to AKH-I from Locusta migratoria: assessment of biological potencies in vivo and in vitro

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu1 and Leu2 (Gly1a–AKH-I) or Leu2 and Asn3 (Gly2a–AKH-I and Gly2ab–AKH-I). Other modified hormones with N-terminal extensions were: (Ahx)n–AKH-I (Ahx, aminohexanoic acid); HPP(Ahx)n–AKH-I (HPP, hydroxyphenyl propionate) and Ac(Ahx)n–AKH-I (where n=0–3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu1: Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro. The potency of AKH-I was reduced much more by insertion of glycine between pGlu1 and Leu2, than between Leu2 and Asn3, perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu2 is necessary for activity (i.e., [despGlu]–AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]–AKH-I is active, but has low potency. The potencies of HPP(Ahx)0–3–AKH-I, Ac(Ahx)1–3–AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx)n–AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu1–AKH-I compared with its unblocked analogue. HPP1–AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.

Glutaminyl-cyclase expression in the bovine/porcine hypothalamus and pituitary.

The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post-translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC-immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.

Refined 1·7 Å X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity

The X-ray crystallographic structure of P-30 protein (Onconase) has been solved by multiple isomorphous replacement and the structure has been refined at 1·7 Å resolution to a conventional R-factor of 0·178. The molecular model comprises all 826 non-hydrogen protein atoms, 96 solvent molecules and a sulfate anion that is bound at the activesite. The molecular structure is similar to that of ribonuclease A. The active site cleft is located at the junction of two three-stranded β-sheets and the N-terminal helix. A sulfate anion is non-covalently bound by Lys9, His10, His97, Phe98 and an intermolecular contact involving Lys55′ from a neighboring molecule. The N-terminal pyroglutamyl (Pyr) residue is part of the active site and its O ϵ1 atom forms a hydrogen bond with the Lys9 N ζ. The previously constructed comparative molecular model of P-30 based on ribonuclease A correctly predicted the overall fold of P-30 and the conformation of its active site residues. The model failed to predict the conformation of Pyr1 and the conformation of the two loops following helix α3 and strand β3.

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis

Bacillus subtilis pyrrolidone carboxyl peptidase (Pep) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal l-pyroglutamic acid from peptides, appears to be a tetramer of 25 200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pep from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 m M for l-pyroglutamyl-β-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C α peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNlVCECCMKPCTLSELRQYCP B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C α peptide has also been isolated and its sequence was determined as NAETDLDDPLRN1KLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.

Isolation and primary structure of an amphibian neurotensin

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog ( Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reversephase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl 1) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.

Amino acid sequence of a peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus

A hydrophobic peptide of 71 residues was isolated from lobster sinus gland extracts that prolonged intermolt periods and lowered ecdysteroid titers in juvenile lobsters. Removal of the N-terminal pyroglutamyl residue allowed sequencing of 30 of the first 36 residues. Additional data were obtained from HPLC-purified fragments from endoproteinase cleavages (Lys-C, Glu-C, Arg-C, Asp-N), and carboxypeptidase Y digestion. This is the first reported amino acid sequence of a crustacean molt-inhibiting hormone. This peptide also has significant hyperglycemic activity.

Processing of pro-hormone precursor proteins

Peptide-hormones and other biologically active peptides are synthesized as higher molecular weight precursor proteins (pro-proteins) which must undergo post-translational modification to yield the bioactive peptide(s). These post-translational enzymatic events include limited endoproteolysis and may include other modifications of the generated peptide such as limited exopeptidase digestion, N-terminal acetylation, C-terminal amidation, and formation of N-terminal pyroglutamyl residues (pyrrolation). The secretory vesicle hypothesis, one of the major hypotheses regarding processing, states that the initial endoproteolytic event occurs upon formation of the secretory vesicle (or granule) or within the secretory vesicle from which the bioactive peptides are released. Two different endoproteinases which are likely to be physiologically relevant processing enzymes of pro-atrial natriuretic factor and pro-gonadotropin releasing hormone precursor protein, respectively, have recently been discovered in our laboratory and are discussed as model enzymes in the context of this hypothesis. The results indicate that the precursor protein and its complement of processing enzymes are co-packaged into the secretory granule. Evidence is presented to support the idea that the specific sequence and conformation (secondary structural features) of the processing recognition site within the precursor protein likely contribute in large part to the basis for limited endoproteolysis. In the pro-hormones studied, the recognition site is an extended sequence of five to seven residues which likely exists as a β-turn at the surface of the precursor protein. By extending our results to appropriate protein sequences in the National Biomedical Research Foundation database, we are suggesting that in addition to the doublet of basic amino acids, the primary processing recognition site in pro-hormone precursor proteins often contains a monobasic amino acid or a strongly polar residue (Glu or Asp) in close sequence proximity to the doublet of basic residues.

Sequences of gastrins purified from a single antrum of dog and of goat

Heptadecapeptide gastrins (G17) have been purified and sequenced from a variety of species. However, progastrin (G34) sequences have been determined only for pig and human from purified peptides and for rat from cDNA. Since G34 in most species accounts for only ∼5% of total antral gastrin, micropurification techniques must be employed to avoid the need for large quantities of antral tissue. Efficient purification methodology yielded 1.5 and 1.3 nmol of G34 from the antrum of a single goat and of a single dog, respectively. The N-terminal pyroglutamyl residues were enzymatically removed and the peptides were sequenced through to the proximity of their COOH-termini. The COOH-terminal sequences of goat and dog G34 were confirmed by sequencing the corresponding deblocked G17 from each animal. The previously published dog G17 sequence was shown to be incorrect. The sequences for dog and goat G34 are: |Dog <ELGLQ G PP QL VADLSKKQGPW M EEEEAAYGWMDF# |Goat <ELGLQ D PP HM VADLSKKQGPW V EEEEAAYGWMDF# Dog and goat gastrins differ in 3 sites in the 17 amino acid NH 2-terminus and only a single site in G17 (the sites of differences are underlined). The ratio for sulfated to non-sulfated antral G17 is 9:1 for the goat and 1:9 for the dog.

Evidence for regulation of a thyrotropin-releasing hormone degradation pathway in GH3 cells.

GH3 cells, cloned from a rat anterior pituitary tumor, synthesize and secrete PRL in response to TRH. One of the pathways of TRH degradation is removal of the N-terminal pyroglutamyl residue catalyzed by pyroglutamyl peptide hydrolase (PPH; EC 3.4.11.8). We recently described the synthesis and properties of 5-oxoprolinal, a specific and potent (Ki = 26 nM) inhibitor of PPH. The effect of long term exposure of GH3 cells to 5-oxoprolinal on PPH activity was studied by incubating cells with inhibitor for 3 days, harvesting, washing to remove inhibitor, and assaying for PPH. Unexpectedly, we found a marked (300%) increase in PPH activity. This effect was dependent on the concentration of 5-oxoprolinal (EC50 = 10(-7) M) and was time dependent, with a rapid increase in enzyme activity occurring during the first 24 h. Cycloheximide did not block the increase. The results suggest that the activity of PPH in GH3 cells is subject to complex regulatory mechanisms.

Pyroglutamyl diazomethyl ketone: Potent inhibitor of mammalian pyroglutamyl peptide hydrolase

Pyroglutamyl peptide hydrolase (EC 3.4.11.8), a cysteine protease, cleaves the N-terminal pyroglutamyl residue from pyroglutamyl peptides such as thyrotropin releasing hormone. Pyroglutamyl diazomethyl ketone was synthesized as an active site directed inhibitor. Preincubation of the partially purified bovine brain enzyme with nanomolar concentrations of inhibitor produced rapid inactivation. Inhibitor concentrations five orders of magnitude higher did not inactivate other exo- and endopeptidases. A dose of 0.1 mg/kg administered intraperitoneally to mice totally inactivated the enzyme in all tissues studied including brain. Pyroglutamyl diazomethyl ketone should be of value in studies on the physiological role of this enzyme in the metabolism of pyroglutamyl-containing peptides.

5-Oxoprolinal: transition-state aldehyde inhibitor of pyroglutamyl-peptide hydrolase

Pyroglutamyl-peptide hydrolase (EC 3.4.11.8) removes the N-terminal pyroglutamyl residue from pyroglutamyl-containing peptides such as thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), neurotensin, and bombesin. The aldehyde analogue of pyroglutamate, 5-oxoprolinal, was synthesized as an active site directed transition-state inhibitor of the enzyme. 5-Oxoprolinal was found to be a potent (Ki = 26 nM) and specific competitive inhibitor of pyroglutamyl-peptide hydrolase. Other aldehydes tested inhibited the enzyme only weakly or not at all. 5-Oxoprolinal blocked the degradation of LH-RH by purified pyroglutamyl-peptide hydrolase. The inhibitor, when injected into mice, inhibited the enzyme after 10 and 30 min. 5-Oxoprolinal should be of value in studies probing the biological significance of pyroglutamyl-peptide hydrolase.

Neurotensin inhibition of gastric exocrine secretions in the cat.

The gastric exocrine inhibitory activities of neurotensin were characterized in conscious cats prepared with gastric fistulae. Neurotensin was a potent inhibitor of pentagastrin-stimulated pepsin secretion (ID50, approx. 0.3 mumol . kg-1 . h-1) but was approximately 60 times less potent against acid secretion. Neurotensin did not significantly reduce submaximal histamine-stimulated acid or pepsin secretions. the total 2 h acid and pepsin outputs in response to insulin-hypoglycaemia were not reduced by neurotensin, although the peak 15-min outputs were reduced. The reduction in peak secretion was possibly related to neurotensin antagonism of the ability of insulin to lower blood glucose concentrations. Neurotensin alone was not hyperglycaemic when given as an intravenous infusion. Two C-terminal fragments of neurotensin, the dodecapeptide and nonapeptide, inhibited pentagastrin-stimulated pepsin secretion, but were less potent than neurotensin. The observations with the C-terminal fragments indicate that the major determinants of gastric exocrine inhibitory activity of neurotensin reside in its C-terminal; this agrees with observations on other biological activities of neurotensin. The reduced potency of the dodecapeptide indicates the importance of the N-terminal pyroglutamyl residue for full gastric exocrine inhibitory activity.