BIOCHEMICAL-PROPERTIES OF DIPEPTIDYL PEPTIDASE-III PURIFIED FROM PORCINE SKELETAL-MUSCLE

Dipeptidyl peptidase III (EC 3.4.14.4) was purified from porcine skele tal muscle by ammonium sulfate fractionation and by chromatography and rechromatography on a Biosept-DEAE HPLC column. The enzyme was purifi ed 2329-fold with a 19% recovery. The relative molecular mass was esti mated to be 82 000 Da by SDS-PAGE and the maximum activity was reached at pH 8.0 and 40 degrees C. The enzyme had a wider range of hydrolysi s on peptide substrates than over dipeptidyl-AMC derivatives, but with out any endopeptidase activity. On the basis of the kinetic parameters and the substrates studied, the tetrapeptide Gly-Gly-Phe-Leu (des-Tyr 1 Leu-enkephalin) showed the best affinity for DPP III. The serine pep tidase inhibitor 3,4-DCI, along with the cysteine protease inhibitors p-CMB and DTNB were potent inhibitors of DPP III. Chelating and reduci ng agents also affected considerably the enzyme activity. Co2+ was pro ved to markedly increase the enzyme activity. Zn2+, Fe2+, Cu2+, Cd2+, and Hg2+ inhibited the activity, while Ca2+ and Mg2+ had no effect. Th e dipeptide Tyr-Tyr exerted an important inhibitory effect on DPP III activity.