A study of a highly specific pyroglutamyl aminopeptidase type-II from the membrane fraction of bovine brain

Gallagher, Seán P.; O’Connor, Brendan

doi:10.1016/s1357-2725(97)00074-5

Cited by 10 publications

(18 citation statements)

References 40 publications

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“…This compared with the value of 3.4 Ϯ 0.7 M (n ϭ 5) which we published recently for the discontinuous fluorometric assay (32). The observed hydrolysis and K i value obtained for Glp-Phe-ProNH 2 are consistent with previous reports for TRH-DE purified from bovine brain (10,31). V max /K m values (Table II) were calculated assuming that the K i values for these peptides correspond to Michaelis constants (40).…”

Section: Hplc Analysis Of the Peptidesupporting

confidence: 89%

“…Indications that this substitution would improve inhibitor binding were deduced from comparison of the kinetic parameters obtained for TRH, TRHAMC, and TRH-OH (Table II). The respective K m values for these peptides were consistent with those reported previously (10). Based on the value of K m for TRHAMC, Gallagher and O'Connor (10) suggested that TRH-DE has a preference for large hydrophobic groups, such as AMC, at the carboxyl terminus of substrates.…”

Section: Table Isupporting

confidence: 87%

“…Several investigators previously examined the ability of TRH derivatives, with modifications to TRH's COOH-and/or NH 2 -terminal residues, to act as substrates or inhibitors of TRH-DE (6,8,10,30). None was found to inhibit TRH-DE activity to any significant extent.…”

mentioning

confidence: 99%

“…None was found to inhibit TRH-DE activity to any significant extent. Until recently, a histidine residue in the P 1 Ј position was thought to be essential for TRH-DE activity (5,8,22), yet it has now been shown that the naturally occurring TRH-like peptide Glp-Phe-ProNH 2 is a substrate for bovine brain TRH-DE (10,31).…”

mentioning

confidence: 99%

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Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin-releasing Hormone-degrading Ectoenzyme for Thyrotropin-releasing Hormone-like Peptides

Kelly¹,

Slator²,

Tipton³

et al. 2000

Journal of Biological Chemistry

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Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system.Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P 1 ) residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P 1 residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K i values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P 1 residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, L-pyroglutamyl-L-asparaginyl-L-prolineamide (K i ‫؍‬ 17.5 M) and Glp-AsnPro-7-amido-4-methyl coumarin (K i ‫؍‬ 0.97 M). Thyrotropin-releasinghormone-degrading ectoenzyme (TRH-DE) 1 (EC 3.4.19.6) is a type II cell surface peptidase located on synaptosomal membranes in the central nervous system (1Ϫ4). TRH-DE catalyzes the hydrolysis of the Glp-His bond in thyrotropin-releasing hormone (TRH), a tripeptide with the amino acid sequence L-pyroglutamyl-L-histidyl-L-prolineamide (Glp-His-ProNH 2 ) (5Ϫ10). This enzyme is strategically placed to play a significant role in extracellular inactivation of TRH, and current evidence strongly indicates that TRH-DE is the principal enzyme responsible for terminating the actions of neuronally released TRH (11Ϫ14).Although first recognized as a hypothalamic regulatory hormone, TRH is now believed to function as a neurotransmitter and/or neuromodulator within the central nervous system (15, 16) where it displays a broad spectrum of stimulatory actions independent of its neuroendocrine functions (15Ϫ17). Based on its central nervous system effects, TRH has been found to have potential use in the treatment of brain and spinal injury (18,19) and several central nervous system disorders, including spinocerebellar degeneration, cognitive deficits, and spinal cord pain transmission (16,17). The mechanisms by which TRH improves these conditions are not fully elucidated but appear to involve the potentiation by TRH of other neurotransmitter systems. Despite its promise, the use of TRH as a therapeutic agent is critically undermined by its susceptibility to proteolytic degradation (20).Compounds that potently and selectively inhibit TRH-DE may be use...

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Section: Hplc Analysis Of the Peptidesupporting

confidence: 89%

Section: Table Isupporting

confidence: 87%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin-releasing Hormone-degrading Ectoenzyme for Thyrotropin-releasing Hormone-like Peptides

Kelly¹,

Slator²,

Tipton³

et al. 2000

Journal of Biological Chemistry

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“…Schomburg et al 18) and Gallagher et al 19) reported that K m values for TRH hydrolysis by recombinant human PAP-II and purified bovine brain PAP-II were 0.030 and 0.040 mM, respectively. These K m values are similar to the K m values by the PAP-Is in our study, indicating that the affinity toward TRH is equivalent between PAP-I and PAP-II.…”

Section: Discussionmentioning

confidence: 99%

Marginal Involvement of Pyroglutamyl Aminopeptidase I in Metabolism of Thyrotropin-Releasing Hormone in Rat Brain

Abe

Fukuda

Tokui

2004

Biological & Pharmaceutical Bulletin

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Thyrotropin-releasing hormone (TRH) is a peptide hormone released from hypothalamic tissue and stimulates the release of thyroid-stimulating hormone (thyrotropin) from the mammalian anterior pituitary by binding to the TRH receptor on the cell surface of the pituitary.1,2) TRH is a tripeptide with the amino acid sequence L-pyroglutamyl-L-histidyl-L-prorineamide (L-pGlu-His-Pro-NH 2 ). The wide distribution of TRH throughout the central nervous system and the findings of various biochemical, pharmacological and behavioral studies strongly suggest that TRH likely acts as a neuromodulator/neurotransmitter in extrahypothalamic brain areas. 2,3)Current evidence strongly indicates that pyroglutamyl aminopeptidase II EC 3.4.19.6) is the principal enzyme responsible for terminating the actions of neuronally released TRH by removing the L-pGlu residue.4-6) PAP-II is a membrane-anchored zinc-metalloenzyme primarily located in the central nervous system and is known as a highly specific enzyme for TRH and very closely related compounds. 1,7)Pyroglutamyl aminopeptidase I (PAP-I, EC 3.4.19.3), a typical cytosolic cysteine peptidase, also can hydrolyze TRH as well as bioactive neuropeptides such as luliberin (LH-RH) and neurotensin due to its broad substrate specificity. [8][9][10] But the cytosolic peptidases are generally considered to have no role in the metabolism of these bioactive peptides, because neuropeptides are commonly hydrolyzed by ectoenzymes localized on the membrane of cells with the active site facing the extracellular space or by lysosomal peptidases after endocytosis.1,11) Nevertheless, the contribution of cytosolic PAP-I to the hydrolysis of TRH in vivo is controversial. Charli et al. 6) reported that pGlu diazomethyl ketone, a PAP-I inhibitor, did not enhance TRH levels in the brain in vivo or in vitro, but Faivre-Bauman et al. 12) observed increased levels of TRH when primary cultures of hypothalamic cells were treated with Z-Gly-ProCHN 2 , also known as a PAP-I inhibitor.Recently we expressed the functional forms of rat, mouse and human PAP-Is by Escherichia coli as a single protein and obtained an antibody raised against rat recombinant PAP-I. 13)Using these preparations, we showed that L-pGlu p-nitroanilide (L-pGlu-pNA) is a good non-peptide synthetic substrate for recombinant PAP-Is and is hydrolyzed solely by cytosolic PAP-I from the immunoprecipitation study using the antibody.13) We also demonstrated that the liver and kidney show relatively high PAP-I activities compared with other tissues tested.13) More recently we found that PAP-I is involved in the hydrolysis of some xenobiotic compounds having an LpGlu or L-pGlu-related structure, now under development in our company. 14,15) In this study, by using the recombinant PAP-Is and the antibody against rat PAP-I, we investigated the possible contribution of PAP-I to the degradation of TRH in rats based on biochemical experiments in brain subcellular fractions and immunohistochemical localization of PAP-I in the brain as well as the liver and kid...

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

Kelly

Slator

Tipton

et al. 1999

Analytical Biochemistry

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A study of a highly specific pyroglutamyl aminopeptidase type-II from the membrane fraction of bovine brain

Cited by 10 publications

References 40 publications

Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin-releasing Hormone-degrading Ectoenzyme for Thyrotropin-releasing Hormone-like Peptides

Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin-releasing Hormone-degrading Ectoenzyme for Thyrotropin-releasing Hormone-like Peptides

Marginal Involvement of Pyroglutamyl Aminopeptidase I in Metabolism of Thyrotropin-Releasing Hormone in Rat Brain

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

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