Purification and characterization of the thyrotropin‐releasing hormone (TRH)‐degrading serum enzyme and its identification as a product of liver origin

Abstract

Previous biochemical studies have indicated that the membrane-bound thyrotropin-releasing hormone (TRH)-degrading enzyme (TRH-DE) from brain and liver and the serum TRH-DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH-DE into the culture medium could also be demonstrated. The trypsin-solubilized particulate liver TRH-DE and the serum TRH-DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH-DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting. Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.Keywords: TRH-degrading enzyme (TRH-DE); serum; liver; brain; characterization.The signal substance thyrotropin-releasing hormone (TRH), a hypothalamic hypophysiotropic neuropeptide hormone (reviewed in [1,2]) and a peptidergic neurotransmitter/neuromodulator (reviewed in [3,4]), is known to be rapidly inactivated by the brain TRH-degrading enzyme (TRH-DE), an ectoenzyme located on the surface of neuronal cells, as well as by the soluble serum TRH-DE. The highest activity of the membrane-bound TRH-DE is found in brain and significant activities are also detected in retina, lung and liver but not in other tissues such as heart, kidney and muscle [5][6][7]. Because the membrane-bound brain TRH-DE and the serum TRH-DE exhibit the same extraordinary high degree of substrate specificity and identical enzyme-chemical characteristics [8][9][10][11][12][13][14] it has been suggested that both enzymes are derived from the same gene.Based on the observation that the developmental pattern of the particulate liver TRH-DE and the serum TRH-DE are almost identical it has been proposed that the serum TRH-DE, like most serum enzymes and proteins, might be of liver origin [9,15]. This interpretation was supported by the findings that the activities of the particulate liver enzyme, like the serum enzyme [16][17][18], is also regulated by thyroid h...

Thyrotrophin‐Releasing Hormone Decreases Feeding and Increases Body Temperature, Activity and Oxygen Consumption in Siberian Hamsters

“…This short‐acting effect of TRH is likely to be due to its relatively rapid metabolism both centrally and peripherally; for example its half‐life has been estimated to be 2 min in mouse brain following i.c.v. infusion (30) and is likely to be as rapid in the circulation (31).…”

Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts

“…Rather, a unique membrane-bound type II PGP, highly specific for TRH [28], called TRH-degrading enzyme, inactivates TRH at the cell surface [29]. A soluble form of this enzyme is shed from the cell surface which regulates TRH levels in the plasma [30], and is the primary TRH-degrading activity in healthy serum and plasma [31]. Type I PGPs do not play a significant role in the normal degradation of TRH in vivo [32].…”

Cell-Surface Peptidases

“…Pyroglutamyl-peptidase II is a strict substrate-specific enzyme: It liberates the terminal pyroglutamic acid from thyrotropin-releasing hormone (TRH); other pyroglutaminyl peptides such as gonadoliberin or neurotensin are not attacked (Bauer, 1987(Bauer, , 1994. The enzyme is found in particular in the brain (neurons), anterior pituitary (mammotrops), retina, lung, and liver (Bauer et al, 1990); also a soluble serum, obviously shedded form exists (Schmitmeier et al, 2002). The extraordinarily high degree of substrate specificity and the restricted localization indicate that pyroglutamyl-peptidase II has a very specialized function as an inactivation enzyme for TRH as a neurotransmitter in the brain and as a hypothalamic releasing hormone in the circulation (Bauer 1994;Schomburg et al, 1999).…”