BOVINE BRAIN PYROGLUTAMYL AMINOPEPTIDASE (TYPE-1) - PURIFICATION AND CHARACTERIZATION OF A NEUROPEPTIDE-INACTIVATING PEPTIDASE

Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble , broad specificity aminopeptidase, capable of removing the pyroglutam ic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study wa s to undertake, for the first time, the complete purification and char acterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield o f 6.6%. The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturi ng conditions via SDS PAGE (24,000 Da), suggesting that the enzyme exi sts as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. Strong inhibi tion of PAP activity was observed with a number of different agents, i ncluding transition metal ions, sulphydryl-blocking agents and 2-pyrro lidone (a pGlu analog). A broad pyroglutamyl substrate specificity, wh ich excludes substrates commencing with the pGlu-Pro bond, was also de monstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, th e soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3). C opyright (C) 1996 Elsevier Science Ltd