Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme

Abstract

The membrane-bound enzyme which catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH,) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200 000-fold, with an overall yield of 20%, by conventional chromatographic methods.The enzyme preparation appeared to be electrophoretically homogenous since SDSPAGE analysis revealed a single band with a molecular mass of 116000 Da. By gel-filtration chromatography, a molecular mass of 230000 Da was estimated, suggesting that the enzyme consists of two identical subunits.The enzyme could be identified as a glycoprotein by lectin-binding analysis and by the reduction of the molecular mass to 97000 Da upon treatment of the denatured enzyme with endoglycosidase-F/N-glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymic activity.In addition to TRH, the enzyme also hydrolyzed ~-5-oxoprolyl-~-naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis-Menten kinetics, but considerable differences in the respective K , and V,,, values were noticed. TRH (thyrotropin releasing hormone) not only acts as a hypothalamic hypophysiotrophic hormone (for review, see [ 1 and 21) but also elicits a variety of effects in the central nervous system [3 -51 indicating that in extrahypothalamic brain areas this peptide may possibly act as a neuromodulator or neurotransmitter. Such functions imply the existence of a highly efficient inactivation system to clear the target site for transmission of the next signal. There is considerable evidence that the inactivation of neuronally released TRH is catalyzed by a particular enzyme (for review, see [6]) that, [7 -91 similar to the TRH-degrading serum enzyme [lo], exhibits a high degree of substrate specificity. This peptidase is found in synaptosomal [7] and adenohypophyseal [ l l ] membrane fractions; in brain cultures it is only found on the surface of neuronal cells and not on glial cells [12-141. In the pituitary it is localized preferentially on lactotrophs [13] ; recent studies demonstrated that the activity of the adenohypophyseal enzyme is regulated by estradiol [14] and is stringently controlled by thyroid hormones [ 16 -181, suggesting that this enzyme itself might serve regulatory functions. To further our understanding of the biological function of this enzyme, we were interested in purifying it to electrophoretic homogeneity as a basis to develop new tools for further studies.Correspondence to K. Bauer, Max-Planck-Institut fur experimentelle Endokrinologie, Feodor-Lynen-Str. 7, D-30625 Hannover, GermanyAbbreviations. P-NA, 8-naphthylamide; TRH, thyrotropin-releasing hormone ; TRF, thyrotropin-releasing factor.Enzyme. Phospholipidase type I11 (EC 3.1.4.3). MATERIALS AND METHODS MaterialsTrypsin, chymotrypsin, proteinase K and endoglycosida...

Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II

“…These data are consistent with the soluble nature of PPI, the addition of a PPI inhibitor in the assay buffer, and the very low catalytic turnover that PPII displays with ϽGlu-␤NA as substrate (22). None of PPII mutants had detectable activity (not shown).…”

“…Rat and human PPII cDNAs encode sequences with a high degree of conservation (19,20). Purification of the brain enzyme indicates that it is a glycoprotein composed of two identical subunits with a molecular mass of 230 kDa when solubilized with trypsin (21,22).…”

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

“…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). 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).…”

“…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).…”

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

“…2,4,5) Higher specific activity levels of PAP-II are found in the cerebral cortex, olfactory bulb, hippocampus and cerebellum, but lower levels are present in the spinal cord. 6,20,21) Heuer et al 3) have demonstrated that basal PAP-II activity in the pituitary is very low, but the activity and the mRNA levels of adenohypophyseal PAP-II are dramatically increased by thyroid hormones, whereas PAP-II activities in other brain regions are not, showing that adenohypophyseal PAP-II activity is stringently regulated by thyroid hormones.…”