Delineation of a Particulate Thyrotropin‐Releasing Hormone‐Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

Abstract: The degradation of thyrotropin‐releasing hormone in rat brain homogenates was studied in the presence of N‐benzyloxycarbonyl‐prolyl‐prolinal and pyroglutamyl diazomethyl ketone, specific and potent active‐site‐directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin‐releasing hormone‐degrading enzyme(s). Reports of a thyrotropin‐releasing hormone‐degrading enzyme with narrow specificity that cleaves the pGlu‐His bond of this tripeptide led us to develop a coupled assay using pGlu‐His‐Pro‐2NA as the substrate to measure this activity. Cleavage of the pGlu‐His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl‐peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o‐phenanthroline. The particulate pyroglutamyl‐peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin‐releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu‐His bond of thyrotropin‐releasing hormone but is distinct from pyroglutamyl peptide hydrolase.