Since the initial isolation of pyroglutamyl aminopeptidase (PAP) from a strain of Pseudomonas fluorescens by Doolittle and Armentrout in 1968, similar enzyme activities have been isolated and characterised from a multitude of prokaryotic and eukaryotic sources Studies on eukaryotic PAPs have been done mainly m mammals, typically with a view to elucidating the potential role of this class of enzymes in the catabolism of various pGlu-terminating peptides, including neuropeptides, in vivo. The central aim of this study was to undertake the complete purification and characterisation of a PAP activity observed within the soluble or cytosolic fraction of bovine whole brain Several workers have previously desenbed the purification and characterisation of soluble PAP activities from different mammalian tissues including guinea-pig and human brain. However, other than some minor details furnished by earlier studies, little was previously known of the bovine brain activity. A combination of different chromatographic methodologies subsequently generated a soluble PAP activity with a total active yield of 6 6% which had been purified to near homogeneity, as judged by SDS PAGE and silver staining techniques. The unstable nature of the purified enzyme in dilute solution was very apparent, prompting the usage of 0 5%w/v BSA to stabilise PAP activity dunng both assay and storage Characterisation of this enzyme activity subsequently revealed a number of interesting results, many of which compared well with findings previously reported for soluble PAP activities examined m other sources In addition to a predominantly cytosolic subcellular location, this enzyme was found to exhibit a low relative molecular mass Gel-filtration chromatography revealed a native molecular mass of approximately 23,700 dal tons, a value which compares well with that obtained for the enzyme under denatunng conditions via SDS PAGE (22,450 daltons), supporting the likelihood that the soluble bovine brain PAP exists as a monomer. A pH optimum of 8 5-9 0, as determined with pGlu-MCA at 37°C, was also demonstrated for this enzyme, whilst the expression of PAP activity exhibited an absolute requirement for the presence of a disulphide bond-reducmg agent such as DTT, suggesting the participation of active site thiol groups in enzyme activity (i.e. a thiol protease) Strong inhibition of punfied PAP activity was observed with a number of different agents which included the transition metal ions Hg2+, Cu2+, Zn2+ and Cd2+, the sulphydryl-blocking agents lodoacetate, 2-iodoacetamide, PCMB and N-ethylmaleimide and the reversible inhibitor 2-pyrrolidone Senne protease inhibitors and metal chelating agents (with the exception of 1,10-phenanthroline) as well as the compounds bacitracin puromycin and bestatin had no effect on enzyme activity. The cleavage of the N-terminal pGlu residue from a wide range of pyroglutamyl substrates including TRH, acid TRH, pGIu-His-Pro-MCA (a TRH analog), bombesin and neurotensin was clearly demonstrated for the soluble bovine brain PAP activity N-termmal pGlu cleavage of eledoisin and LHRH could not be detected however Whereas this was expected for eledoisin, a substrate which commences with the sequence pGlu-Pro, such a finding for LHRH was quite unexpected Subsequent kinetic analysis also revealed that (he punfied PAP activity displays Km and K, values withm the lower micromolar range for a number of these substrates (TRH, acid TRH, LHRH, pGlu-MCA, pGlu- BNA and pGIu-His-Pro-MCA) indicative of a strong enzyme-substrate interaction. In addition, all of the pGlu-peptides for which Kj values were estimated proved to be competitive inhibitors. Based on a comparison of these findings with those reported for soluble PAP activities in other mammalian tissues, the soluble PAP enzyme activity observed in bovine whole bram can tentatively be classified as a pyroglutamyl ammopeptidase type-1 or PAP-I (EC 3 4 19 3)