Characterization of a Thyroliberin‐Degrading Serum Enzyme Catalyzing the Hydrolysis of Thyroliberin at the Pyroglutamyl‐Histidine Bond

The enzymatic hydrolysis of thyroliberin (< Glu‐His‐Pro‐NH 2 ) by serum enzymes can be preferentially inhibited by 1 mM diisopropylfluorophosphate. Under such conditions, pyroglutamic acid and His‐Pro‐NH 2 are formed as the only enzymatic split products. As a secondary metabolite, histidylproline diketopiperazine is formed non‐enzymatically by intramolecular condensation of the dipeptide amide. Due to their differences in charge, the enzymatically formed split products can easily be separated from thyroliberin by ion‐exchange chromatography on cellulose‐phosphate paper and thus, by using radioactively labelled thyroliberin as tracer, highly sensitive tests could be developed for the quantitative determination of the thyroliberin‐degrading enzymatic activity. By gel filtration, the thyroliberin‐degrading serum enzyme could be partially purified and separated from two enzymes which catalyze the hydrolysis of pyroglutamyl β‐naphthylamide, the substrate of the known pyroglutamate aminopeptidases. For the serum tested, the extremely low activities of the pyroglutamate aminopeptidases did not contribute to the observed rapid degradation of thyroliberin. On the other hand, pyroglutamyl β‐naphthylamide was not effectively hydrolyzed by the thyroliberin‐degrading serum enzyme. Furthermore, in contrast to the pyroglutamate aminopeptidases, which are activated by EDTA and dithiothreitol, the thyroliberin‐degrading serum enzyme was inhibited by these agents. This enzyme, having an approximate molecular weight of 260000, is also effectively inhibited by other chelating agents, by heavy metal ions and by p ‐chloromercuribenzoate but not by 2–iodoacetamide and N ‐ethylmaleimide or by serine protease inhibitors. When tested in vitro , the activity of the thyroliberin‐degrading serum enzyme was not affected by thyroid hormones, steroids or adenohypophyseal hormones. These results demonstrate that the degradation of thyroliberin at the pyroglutamyl‐histidine bond is catalyzed by a serum enzyme which exhibits unique enzyme‐chemical characteristics distinctly different from those of the known pyroglutamate aminopeptidases catalyzing the same reaction.