Cytosolic bovine brain prolyl oligopeptidase was purified from whole brain using ammonium suphate precipitation and chnitnjtography with DEAE sepharose, S200 gel filtration, chromatofocusmg and phenyl sepharose An overall recovery of 23% and purification factor of 253 was achieved. The relative molecular mass of the brain PO, determined by gel filtration chromatography, was found to be 69 5 kDa This was confirmed by SDS PAGE The purified enzyme was relatively unstable under assay conditions However the presence of 0 5% w/v BSA improved its stability and the assay linearity Activity was also inhibited strongly by solvents with DMF being the most inhibitory DMSO was found to be the optimal solvent in terms of enzyme activity and substrate solubility Optimal enzyme activity was observed at 37°C with complete inactivation occurring at temperatures of 50°C or more A pH optimum of 7 4 and a preference for phosphate buffer was found for the enzyme with complete inactivation of activity below pH 4 5 and above pH 10 The brain PO was confirmed to be a serine protease based on its sensitivity to AEBSF The enzyme was also inhibited strongly by some cysteine protease inhibitors such as NEM and DTNB and was activated by DTT Sensitivity to these agents would suggest the presence of a cysteme residue in close proximity to the active site Some divalent metal salts also exerted some inhibitory effects on activity with Hg2+, Cu2+, Cd2+, Co2+ and Ni2+ being the most potent Substrate spcUilcity studies performed on the purified bovine brain, partially purified bovine serum and recombinant Flavobacterium meningosepticum PO activity revealed that this oligopeptidase could hydrolyse a range of proline containing peptides including TRH, LHRH, ADNF-14, substance P, neurotensin, CLIP, a 45 amino acid residue Gly-Pro-Ala polymer and the APP fragment 708-715 N-blocked prolme containing dipeptides, including Z-Pro-Pro-OH were not hydrolysed by any of the enzyme The smallest synthetic sequences hydrolysed were an N-blocked tripeptide and a tetrapeptide The brain, serum and bacterial activities hydrolysed Z-Gly-Pro-MCA, with Km values of 62 5, 14 6 and 38 5(iM respectively The TRH analog pGlu-His-Pro-MCA was also hydrolysed by all three activities with higher Km values of 99 8, 52 1 and 73 5(xM for the brain serum and recombinant Flavobacterium meningosepticum enzyme respectively A number of prolme-containing peptides were also found to competitively inhibit all three activities Of these angiotensins I, II and II were the most potent The TRH analogs, Glu2TRH and Phe1TRH exhibiting the lowest inhibitory potency Inhibition studies performed using a range of PO-specific inhibitors revealed a-ketobenzothiazole to be the most potent inhibitor of bovine brain PO with an IC50 value of 63pM The classical PO inhibitor Z-Pro-prolinal inhibited all three activities with IC50 values of 7-10nM With regard to the majority of inhibitors tested, the brain, serum and bacterial enzymes were similar m their sensitivity However the brain and serum activities were approximately 4000 times less sensitive than the bacterial enzyme to inhibition by the N-blocked dipeptide analog, Z-Phe-Pro-methylketone An investigation into the time course inhibition of bram and bacterial activities by Z-Phe-Alachloromethylketone found that while the bacterial enzyme was completely inhibited by lxlO'5 M of this inhibitor alter 60 minutes, the bram enzyme was completely insensitive to inhibition