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

Kelly, Julie; Slator, Gillian R.; Tipton, Keith F.; Williams, C. H.; Bauer, Karl

doi:10.1006/abio.1999.4276

Cited by 7 publications

(16 citation statements)

References 35 publications

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“…Enzyme Purification-TRH-DE was purified almost 20,000-fold from porcine brain as described previously (32,35). A Coomassie-stained gel of the purified enzyme after SDS-polyacrylamide gel electrophoresis showed one major band, the molecular mass of which was consistent with that of 116 kDa reported previously for TRH-DE (35).…”

supporting

confidence: 66%

“…New and improved TRH-DE assays (32) were employed to investigate the kinetic properties of the peptide library rigorously. The data presented here provide the first illustration of TRH-DE specificity across a library of TRH-like peptides.…”

mentioning

confidence: 99%

“…This highly purified TRH-DE preparation did not contain any of the various peptidase activities tested, including aminopeptidases, carboxypeptidases, and dipeptidyl aminopeptidases, and it was completely devoid of other TRH-degrading enzymes, including pyroglutamyl aminopeptidase I (EC 3.4.21.26) and prolyl oligopeptidase (EC 3.4.19.3) (32). The preparation was found to have (i) a protein concentration of 0.8 mg ml Ϫ1 using a modification of the Lowry method (36) with bovine serum albumin as a standard and (ii) a specific activity of 0.17 unit mg Ϫ1 with TRHAMC as substrate under standard conditions of a continuous assay described previously (32) and outlined below.…”

mentioning

confidence: 99%

“…This preparation did not contain any measurable amount of either dipeptidase or oligopeptidase activity. The specific activity was 17.5 units mg Ϫ1 with Gly-ProAMC as substrate (32).…”

mentioning

confidence: 99%

“…Therefore, those peptides undergoing significant hydrolysis (i.e. those that exhibited rates of hydrolysis Ն 0.5 unit mg We have shown that the amount of AMC formed under these conditions is a quantitative measure of TRHAMC cleavage (32). Initial rates for the hydrolysis of TRHAMC by TRH-DE were determined in triplicate at five different substrate concentrations, both in the absence and presence of at least three concentrations of peptide.…”

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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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confidence: 66%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…This preparation did not contain any measurable amount of either dipeptidase or oligopeptidase activity. The specific activity was 17.5 units mg Ϫ1 with Gly-ProAMC as substrate (32).…”

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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Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review

et al. 2001

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In recent years our research group has developed new alternatives for fluorescence enzymatic determinations. First, we observed that the intrinsic fluorescence of enzymes changes during enzymatic reactions, proportionally to the substrate concentration, avoiding the combination of the enzymatic reaction with a fluorophore-involving reaction. The main disadvantage of this method is that the excitation and emission wavelengths of the enzymes are in the UV region of the spectrum. An alternative to overcome this problem consisted of covalently bonding the enzyme to a fluorophore. In this paper, an overview is given of all of the applications and future developments on both types of alternatives that we have developed. Apart from the analytical characteristics of the methods, we have also reviewed all of the information about mathematical models we have elaborated to date.

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Pyroglutamyl-peptidase II

Bauer

2004

Handbook of Proteolytic Enzymes

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

Cited by 7 publications

References 35 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

Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review

Pyroglutamyl-peptidase II

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