STUDIES ON THE EFFECTS OF INHIBITORS OF TRH-DEGRADING ENZYMES (PROLYL ENDOPEPTIDASE, PYROGLUTAMYL PEPTIDE HYDROLASE).
Item
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Title
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STUDIES ON THE EFFECTS OF INHIBITORS OF TRH-DEGRADING ENZYMES (PROLYL ENDOPEPTIDASE, PYROGLUTAMYL PEPTIDE HYDROLASE).
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Identifier
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AAI8601642
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identifier
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8601642
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Creator
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FRIEDMAN, THEODORE C.
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Contributor
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Sherwin Wilk
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Date
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1985
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Language
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English
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Publisher
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City University of New York.
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Subject
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Health Sciences, Pharmacology
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Abstract
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The effects of several specific inhibitors of enzymes capable of degrading thyrotropin-releasing hormone (TRH; pyroglutamyl-histidyl-prolyl-amide) was studied. These studies were carried out in rat brain, pituitary and serum and in cell culture (GH3 cells). The inhibitors studied were N-benzyloxycarbonyl-prolyl-prolinal (Z-Pro-Prolinal), a specific, potent (Ki = 14 nM), transition-state analog inhibitor of prolyl endopeptidase, 5-oxoprolinal, a newly synthesized transition-state analog inhibitor of pyroglutamyl peptide hydrolase and pyroglutamyl diazomethyl ketone, an inhibitor capable of irreversibly alkylating the active-site cysteine residue of pyroglutamyl peptide hydrolase.;5-Oxoprolinal was synthesized by conversion of pyroglutamate to its methyl ester, reduction of the ester to the alcohol (5-oxoprolinol) by sodium borohydride followed by oxidation of the alcohol to the aldehyde. Kinetic experiments showed that 5-oxoprolinal competitively inhibited thiol-dependent pyroglutamyl peptide hydrolase with a Ki of 26 nM.;Experiments were conducted in male Swiss Albino mice with the three inhibitors. The activity of prolyl endopeptidase was inhibited by more than 85% in homogenates of all tissues studied 30 minutes after intraperitoneal injection of Z-Pro-Prolinal. The in vivo degradation of a prolyl endopeptidase substrate. N-benzyloxycarbonyl-Gly-Pro-Sulfamethoxazole, was blocked after administration of Z-Pro-Prolinal in a dose-and time-dependent manner, indicating inhibition of the enzyme in vivo. 5-Oxoprolinal, when injected into mice, inhibited pyroglutamyl peptide hydrolase after 10 and 30 min however a relatively high dose (50 mg/kg) was needed to achieve this inhibition. Pyroglutamyl diazomethyl ketone was injected into mice and at a dose of 0.1 mg/kg totally inactivated the enzyme in all tissues studied including brain.;It was expected that the combined use of inhibitors to prolyl endopeptidase and pyroglutamyl peptide hydrolase would totally protect TRH from degradation in vitro. However, when tissue homogenates or serum was incubated in the presence of inhibitors but in the absence of metal chelators, substantial TRH degradation was observed. This indicated the presence of another TRH-degrading enzyme(s) distinct from pyroglutamyl peptide hydrolase.;Z-Pro-Prolinal was injected into rats to see if the inhibition of prolyl endopeptidase would raise endogenous TRH levels. TRH levels were found to be significantly elevated in the pituitary 15 minutes after injection of Z-Pro-Prolinal (5 mg/kg).;Experiments were carried out to study the effects of inhibitors in GH3 cells, a cell line cloned from a rat anterior pituitary tumor, which synthesize and secrete prolactin in response to thyrotropin-releasing hormone. (Abstract shortened with permission of author.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
