Pyroglutamyl peptidase from chicken liver: Purification and some properties.

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Abstract
Pyroglutamyl peptidase was purified from chicken liver by a series of column chromatographies on DEAE-Sephadex A-50, PCMB-T Sepharose, Sephadex G-150 and hydroxyapatite. By these procedures, the enzyme was purified about 3800-fold with an activity recovery of 3.6%. The enzyme was most active and stable at pH7-8.2. Mercaptoethanol and EDTA stabilized the enzyme effectively. The enzyme was inactivated by incubation with sodium tetrathionate, PCMB, Ni2+, Cd2+, Zn2+ and Hg2+, but not affected by serine protease inhibitors such as DFP and phenylmethanesulfonyl fluoride (PMSF). Thiolprotease inhibitors, E-64 and Ep-475, were partially inhibitory to the enzyme. The MW of the enzyme was estimated to be 86,000 by the gel filtration method and the isoelectric point was at pH 5.5 as checked by isoelectric focusing method. The enzyme readily hydrolyzed pyroglutamyl-.beta.-naphthylamide (Pyr-2-NNap), Pyr-p-nitroanilide (Pyr-pNA), Pyr-4-methylcoumarinamide (Pyr-MCA) and most of Pyr-amino acids tested, but was only slightly active toward Pyr-D-Ala and completely inert toward Pyr-Pro. Kinetic analysis of the reaction indicated that the latter 2 peptides competitively inhibit the enzyme reaction with Ki values of 7.8 and 13.5 mM, respectively. The Km values for Pyr-derivatives of 2-NNap, MCA, pNA and Ala were 0.7, 0.004, 0.73 and 0.89 mM, respectively. The enzyme split bradykinin potentiator, neurotensin, LHRH, TRH, frog litorin and physalaemin, liberating pyroglutamic acid from their amino termini, but was inactive toward uperolein and the TRH analog, piperidone carbonyl His-Pro-NH2.

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