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- W978047253 abstract "Three new fluonmetnc assays were developed for the detection of pyroglutamyl ammopeptidase type-II (PAPII) activity. Two of these assays are based on hydrolysis of the TRH substrate analogue pGlu-His- Pro-MCA, while the third, a continuous assay, exploits the ability of the enzyme to hydrolyse the substrate pGlu-MCA.Following solubilisation of PAPII from the membrane fraction o f bovme brain, using trypsin, the enzyme was purified 3,000-fold, with an overall yield of 24%, using a range of conventional chromatographic techniques.By gel filtration chromatography, a relative molecular mass of 214,000 Da was determined for the enzyme The purified PAPII was found to be relatively labile. However, the presence of 1% v/v BSA was shown to greatly improve its stability. Optimal enzyme activity was observed at 45 °C A pH optimum of 6 8-7 6 was observed for the enzyme, with rapid inactivation occurring below pH 40 and above pH92.Purified PAPII was strongly inhibited by the transition metals Cd2+, Hg2+ and Zn2+, while activation was observed m the presence of Co2+. The enzyme was identified as a metallopeptidase 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, including Zn2+, Ni2+ and Co2+ Inhibition by cysteine protease inhibitors and activators and by the senne protease inhibitor AEBSF, was also observed.Substrate specificity studies revealed that, with the exception of pGlu-Phe-Pro-NH2, pGlu-MCA and pGlu-BNA, the purified enzyme cleaves N-terminal pyroglutamic acid from only tn- and tetrapeptides with a His residue m the penultimate position. A number of N-terminal pyroglutamyl peptides of varying length were shown to competitively inhibit the enzyme. Of these, LHRH and LHRH 1-5, although not substrates for the enzyme, were found to be potent inhibitors, with Kj values of 8|xM and 11 (iM respectively." @default.
- W978047253 created "2016-06-24" @default.
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- W978047253 date "1996-01-01" @default.
- W978047253 modified "2023-09-27" @default.
- W978047253 title "The purification and characterisation of a membrane bound pyroglutamyl aminopeptidase type-II from bovine brain" @default.
- W978047253 hasPublicationYear "1996" @default.
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