Human Lung Post-Proline Endopeptidase: Purification and Action on Vasoactive Peptides

Post-proline endopeptidase (PPE, EC 3.4.21.26) was purified 3,450 times from human lung. PPE was routinely assayed with the artificial substrate, carbobenzoxy-glycyl- L-prolyl-p-nitroanilide (Z-Gly-Pro-pNA). The pH optimum was 7.4, and the M(r) was 77,000. Thiol blocking agents were strongly inhibitory but serine blocking agents were not inhibitory. No metal ions were required for activity, but heavy metal ions such as Hg^2+, Cu^2+, Cd^2+, and Zn^2+ completely inactivated the enzyme. Both dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) were required to stabilize PPE activity. Michaelis constant values for Z-Gly-Pro-pNA and carbobenzoxy-glycyl-L-prolyl-2-naphthylamide were 0.36 and 0.10 mmol/1, respectively. PPE cleaved vasoactive peptides including bradykinin (BK) and des- (Arg^9)-BK (Pro^3-Gly^4 and Pro^7-Phe^8 bonds), angiotensins I and II (Pro^7-Phe^8 bond), substance P (Pro^4-Gln^5 bond), and oxytocin (Pro^7-Leu^8 bond). Each of these peptides inhibited PPE-catalyzed hydrolysis of Z-Gly-Pro-pNA competitively. BK had the lowest Kj value (2.35 pmol/1) and oxytocin had the highest Kj value (84.0 pmol/1). PPE was not inhibited by captopril, a potent inhibitor of angiotensin converting enzyme, which also cleaves the Pro^7- Phe^8 bond of BK.