A study of a highly specific pyroglutamyl aminopeptidase type-II from the membrane fraction of bovine brain.

Pyroglutamyl aminopeptidase type-II is reported to be a highly specific, membrane-bound neuropeptidase, which has the ability to hydrolytically remove the L-pyroglutamyl residue (pGlu) from the N-terminus of thyrotropin releasing hormone (TRH, pGlu-His-Pro-NH2) and closely related tripeptides or tripeptide amides. The primary aim of this study was to purify this enzyme from bovine brain and to compare its characteristics with those previously reported. Following solubilization from the membrane fraction by limited proteolysis with trypsin, the enzyme was purified approximately 3000-fold with a 24% recovery of activity. A native molecular mass of 214,000 Da was determined for the purified enzyme by gel-filtration chromatography. A pH optimum of 6.8-7.6 was observed for the enzyme, with rapid inactivation occurring below pH 4.0 and above pH 9.2. Optimal enzyme activity was observed at 45 degrees C. On the basis of its inhibition, in a time-dependent manner, by metal complexing agents and its subsequent reactivation in the presence of metal ions, the enzyme was identified as a metallopeptidase. Substrate specificity studies revealed that, with the exception of pGlu-Phe-Pro-NH2, pGlu-7-amino-4-methyl-coumarin and pGlu-beta-naphthylamide, the purified enzyme removes N-terminal pGlu from only tri- and tetrapeptides with a histidine residue in the penultimate position. A number of N-terminal pyroglutamyl peptides of varying length were shown to competitively inhibit the enzyme. Of these, luteinizing hormone releasing hormone (LHRH) and LHRH 1-5, although not substrates for the enzyme, were found to be potent inhibitors, with Ki values of 8 and 11 microM, respectively. The study shows that while bovine brain PAPII shares many of the characteristics of PAPII from other mammalian tissues, its substrate specificity is not as narrow as previously reported.