ABSTRACT
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.
ABSTRACT
Pyroglutamyl peptidase II, a highly specific thyrotropin releasing hormone (TRH)-degrading enzyme is found in highest concentration in brain where it is localized to synaptic membranes. Retina contains relatively high concentrations of both immunoreactive TRH and TRH receptors. We report that the specific activity of pyroglutamyl peptidase II in rabbit retinal membranes exceeds that of all non-CNS tissues thus far studied. Nine clonal cell lines were screened for this enzymatic activity. The specific activity of pyroglutamyl peptidase II in Y79 retinoblastoma cells was greater than the highest activity found in other cell lines by approximately one order of magnitude. These studies further support a functional relationship between pyroglutamyl peptidase II and TRH and identify a cell line suitable for studies on the regulation of this enzyme.
