Pyroglutamate Aminopeptidase Activity Research Articles

Removal of N-terminal blocking groups from proteins.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.

Effects of Manduca sexta allatostatin and an analogue on the peach‐potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut

The oral toxicity of the C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analogue δR³δR⁵Manse-AS, where R residues were replaced by their D-isomers, were tested against the peach-potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose-dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³δR⁵Manse-AS, had an estimated LC₅₀ of 0.31 µg/µl diet and was more potent than Manse-AS (estimated LC₅₀ of 0.58 µg/µl diet). At a dose of 0.35 µg δR³δR⁵Manse-AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse-AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half-life of Manse-AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L-like cysteine protease, carboxypeptidase-like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin-like proteases. The half-life of the δR³δR⁵ Manse-AS analogue was enhanced (73 min) with the D-isomers of R appearing to prevent cleavage around the R residues by cathepsin L-like cysteine proteases or from trypsin-like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse-AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies.

Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities

In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different venoms and/or the different abilities of the venoms examined to hydrolyze different peptides during envenomation.

Removal of N‐Terminal Blocking Groups from Proteins

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for N(alpha)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(alpha)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N-terminally blocked proteins using acid-catalyzed hydrolysis or methanolysis, hydrazinolysis, and beta-elimination after acid-catalyzed N-O shift. Alternate protocols describe chemical removal of acetyl and longer-chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.

796 Aβ production following endocytosis of β-amyloid precursor protein (βPP) depends on signal(s) localized in the final 15 amino acids of the C-terminus

The effects of dithiothreitol on pyroglutamate aminopeptidase activity in rat serum, spinal cord, brain, liver, kidney, and adrenal extracts were investigated in vitro. Addition of dithiothreitol to the reaction mixture resulted in noncompetitive inhibition of serum (Ki=2.1±0.3 mM) and brain (Ki=3.2 ± 0.28 mM) enzymes and stimulation of liver, spinal cord, kidney and adrenal enzymes with an increase in Vmax but not Km. The findings of these studies suggest that the properties of Pyroglutamate aminopeptidases vary from tissue to tissue.

A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain

Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH 2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1–3) and the TRH-analogue (methyl-His)-TRH and had a K m of 100 μM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethylmaleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.

Ontogeny of two topologically distinct TRH-degrading pyroglutamate aminopeptidase activities in the rat liver

We recently separated and characterized two topologically distinct pyroglutamate aminopeptidase (PAP) activities in adult rat liver, which convert TRH to cyclo His-Pro (cHP). The liver possesses high-affinity binding sites to the biologically active dipeptide cHP and is thus a potential target tissue for pancreatic TRH and/or its conversion product cHP, and may be a site of TRH conversion and/or inactivation. This report describes the ontogenic development of two liver PAP activities and compares them with that of plasma thyroliberinase. The particulate high-molecular-weight PAP was absent at birth and during the neonatal period, while the soluble, low-molecular-weight PAP was present at all the developmental stages tested. The changes in particulate PAP activity are similar to those in the plasma of age-matched rats. The peculiar age-dependent changes in particulate PAP activity, plus its cellular location, suggest that it has a regulatory role.

Thyroid status regulates particulate but not soluble TRH-degrading pyroglutamate aminopeptidase activity in the rat liver

Rat liver contains two topologically different TRH-degrading pyroglutamate aminopeptidases. The particulate pyroglutamate aminopeptidase, unlike the soluble one, was highly specific for TRH and shared many physico-chemical properties with serum thyroliberinase, which is controlled by thyroid hormones. Both enzymes convert pGlu His Pro NH2 into His Pro NH2; the latter may be cyclized to cyclo His Pro known to possess several biological activities and specific binding sites in liver. The aim of the present study was to determine the effects of thyroid status on the particulate and soluble enzymes activity and gain more insight into their biological role. The regulatory pathway for the particulate pyroglutamate aminopeptidase was found similar to that of serum thyroliberinase: their specific activities decrease in hypothyroid rats and increase in hyperthyroid rats, whereas that of soluble enzyme remains unchanged. We postulate that the particulate pyroglutamate aminopeptidase may be a determining factor in the concentrations of TRH and/or cyclo His Pro reaching liver cells and a possible source for serum thyroliberinase. Taken together, these data suggest that this "converting enzyme" acts as a physiological regulator.

Presence of a Particulate Thyrotropin-Releasing Hormone-Degrading Pyroglutamate Aminopeptidase Activity in Rat Liver

The data of this work describe the presence in the rat liver of a thyrotropin-releasing hormone (TRH)-degrading particulate metal-containing pyroglutamate aminopeptidase of high molecular weight. Following the fractionation of liver homogenate in 0.25 M sucrose, solubilization of the particulate fraction with papain and gel filtration on ACA34, an enzyme activity was detected which converts TRH into pyroglutamic acid and histidyl-proline diketopiperazine (cyclo[His-Pro]; cHP). [L-Histidine-2,5-3H]TRH and [L-proline-2,3-3H]TRH were used as a tracer. Products formed were separated by thin-layer chromatography, localized and quantified by scanning for radioactivity. Enzyme activities were tested using preferential site-directed inhibitors. Particulate pyroglutamate aminopeptidase activity was found to be sensitive to chelating agents. The physicochemical properties of this particulate aminopeptidase were distinct from the soluble pyroglutamate aminopeptidase from the same source. The particulate enzyme shared several similarities with particulate pyroglutamate aminopeptidase from adenohypophysis, brain and with serum enzyme, reported to have narrower specificity for TRH compared to the soluble pyroglutamate aminopeptidase from several tissues. The Km of the gel-filtrated enzyme is 27 microM and the specific activity 306 pmol.min-1.mg protein-1. Although no definite role has been established for this enzyme, it might be a potential determinant of cHP concentrations in liver. Furthermore, cHP is known to possess biological activities and specific binding sites in liver membranes. One of the major sites for TRH breakdown in vivo, the liver, probably represents a target tissue for TRH, especially for pancreatic TRH, and the particulate enzyme involved in the conversion of TRH into cHP may assume a specific function.

Activation/inactivation of rat tissue pyroglutamate aminopeptidase by disulfide bond-reducing agents

The effects of dithiothreitol on pyroglutamate aminopeptidase activity in rat serum, spinal cord, brain, liver, kidney, and adrenal extracts were investigated in vitro . Addition of dithiothreitol to the reaction mixture resulted in noncompetitive inhibition of serum (Ki=2.1±0.3 mM) and brain (Ki=3.2 ± 0.28 mM) enzymes and stimulation of liver, spinal cord, kidney and adrenal enzymes with an increase in V max but not K m. The findings of these studies suggest that the properties of Pyroglutamate aminopeptidases vary from tissue to tissue.

Pyroglutamate aminopeptidase activity in human cerebrospinal fluid decreases with age

The activity of pyroglutamate aminopeptidase, the major enzyme catalyzing thyrotropin-releasing hormone (TRH) metabolism in human CSF, decreased with age. This decrement is not due to age-dependent appearance of any enzyme inhibitor in CSF. The results of these studies underline the importance of using age-matched controls in assessing abnormalities of TRH metabolism in CSF during disease states.

Thyrotropin-releasing hormone metabolism is attenuated in the cerebrospinal fluid of the human neonate

The present study was designed to investigate the ontogeny of thyrotropin-releasing hormone (TRH) metabolism in human cerebrospinal fluid (CFS). The activity of pyroglutamate aminopeptidase (EC 3.4.11.8), the major enzyme catalyzing TRH metabolism in human CSF, was measured in CSF of 11 premature infants (gestational age, 29–39 weeks; birth weight, 1774 ± 274 g), 8 newborn infants (term delivery; birth weight, 3648 ± 240 g), and 11 adults (mean age, 29.6 ± 1.5 years). Pyroglutamate aminopeptidase activity in CSF of premature and newborn infants was significantly lower (p < 0.05) than that of adult CSF. These observed differences in the enzymatic activities were not due to changes in the affinity of the enzyme for its substrate TRH or the presence of enzyme inhibitor(s)/stimulator(s).

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in human cerebrospinal fluid

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone and pyroglutamate aminopeptidase activity was examined in the CSF of human and a number of other mammalian species. Cyclo(His-Pro)-like immunoreactivity was present in the CSF of all species examined, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentration in CSF had no significant correlation with CSF TRH or pyroglutamate aminopeptidase.

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea-pig brain.

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea-pig brain by papain treatment. This activity was further purified 3560-fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid-thyroliberin, the luliberin N-terminal tripeptide (Glp-His-Trp) and, only slightly, Glp-His-Gly. No hydrolysis was observed with dipeptides containing N-terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. A Km value of 40 microM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 microM.

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and pyroglutamate aminopeptidase activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with pyroglutamate aminopeptidase activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.

Histidyl-proline diketopiperazine cyclo (His-Pro): Measurement by radioimmunoassay in human blood in normal subjects and in patients with hyper- and hypothyroidism

An extraction procedure for cyclo (His-Pro) in human blood has been developed, employing sequential extraction with 90% methanol and 0.3 M perchloric acid. Chromatographic elution profiles of immunoreactive cyclo (His-Pro) in blood extracts measured by radioimmunoassay corresponded precisely to those of { 3H} cyclo (His-Pro) following separation with Sephadex G-25, DEAE-cellulose, and SP-Sephadex columns, and also HPLC. Concentrations of cyclo (His-Pro) in normal human subjects (5073 ± 730 fmoles/ml) were approximately 50 times greater than respective TRH concentrations measured in identical samples. Cyclo (His-Pro) concentrations in hypothyroid patients (12373 ± 802 fmoles/ml) were significantly higher than those in normal and those in hyperthyroid patients (5949 ± 566 fmoles/ml). TRH concentrations, by contrast, were similar in all 3 groups. Pyroglutamate aminopeptidase activity which catalyzes the conversion of TRH to cyclo (His-Pro), was identified also in peripheral plasma. The plasma enzyme activity in normal subjects was 76 ± 27 fmoles cyclo (His-Pro) formed/min/ml plasma. This activity was not statistically increased in hypothyroid patients, so that the increased concentrations of cyclo (His-Pro) in hypothyroidism could not be attributed to the enhanced conversion of TRH in the peripheral blood.