An evaluation of the role of a pyroglutamyI peptidase, a post‐proline cleaving enzyme and a post‐proline dipeptidyI amino peptidase, each purified from the soluble fraction of guinea‐pig brain, in the degradation of thyroliberin in vitro

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Abstract
The degradation of thyroliberin (< Glu-His-Pro-NH2) to its component acids by the soluble fraction of guinea pig brain is catalyzed by 4 enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalyzing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalyzed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin, bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolyzed. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalyzed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyze the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalyzed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 .mu.M), the post-proline cleaving enzyme by bacitracin (IC50 = 42 .mu.M) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 .mu.M). Because of their specific inhibitory effects these 3 reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.