Pyroglutamyl peptidase II, a thyrotropin releasing hormone degrading enzyme: purification and specificity studies of the rabbit brain enzyme.

Pyroglutamyl peptidase II (EC 3.4.19.-) a membrane-bound thyrotropin releasing hormone degrading enzyme was purified 5000-fold to apparent homogeneity from the particulate fraction of a rabbit brain homogenate. The enzyme displayed a broad pH optimum in the neutral range. It was inhibited by metal chelators but was unaffected by inhibitors of serine, cysteine or aspartyl proteases. The molecular weight determined on a calibrated Sephadex G-200 column was 230,000. A series of pyroglutamyl peptidyl naphthylamides were synthesized for substrate specificity studies. The K(m) and V(max) of hydrolysis of the naphthylamide (NA) analog of TRH (pGlu-His-Pro-NA) was similar to TRH. The enzyme cleaved pGlu-His-tripeptidyl naphthylamides but did not cleave the pGlu-His bond of the dipeptide pGlu-His-NA or the tetrapeptide pGlu-His-Pro-Ala-NA. The degradation of substrates lacking a chromogenic group was studied by HPLC. The enzyme did not cleave pGlu-His, the pGlu-His decapeptide LHRH, Phe(2)-TRH or the ring opened Glu(1)-TRH. The specificity studies are consistent with the designation of pyroglutamyl peptidase II as the first characterized neuropeptide specific peptidase. On the basis of its localization and specificity, pyroglutamyl peptidase II may control the biological activity of neuronally released TRH.