Specific, high-affinity bradykinin binding by purified porcine kidney post-proline cleaving enzyme.

Post-proline cleaving enzyme (PPCE) was purified from porcine kidney cytosol. The purified enzyme bound [125I-Tyr5]-bradykinin but neither [125I-Tyr1]-kallidin nor [125I-Tyr8]-bradykinin. Scatchard analysis of the data was consistent with a single class of binding sites with a Kassoc = 1.3 +/- 0.1 X 10(8) M-1. The optimal pH for [125I-Tyr5]-bradykinin binding was 6.8. The specificity of binding was evaluated with sixty-seven bradykinin analogs. The catalytic activity of the enzyme was measured with N-benzyloxycarbonyl-Gly-Pro-methylcoumarinyl-7-amide (Z-Gly-Pro-MCA). The optimal pH for hydrolysis of this substrate was broad and centered at 8.3. The apparent Km and Vmax were obtained from Lineweaver and Burk plots and were 4.8 +/- 0.4 X 10(-5) M and 42 +/- 5 mumoles X mg-1 X min-1 respectively. The IC50 values for bradykinin, diisopropylfluorophosphate (DFP), and N-benzyloxycarbonyl-Pro-Prolinal (Z-Pro-Prolinal) to inhibit Z-Gly-Pro-MCA hydrolysis by PPCE were 5.9 +/- 1.4 X 10(-7) M, 8.8 +/- 3.1 X 10(-7) and 7.9 +/- 0.3 X 10(-9) M respectively. Corresponding values for inhibition of [125I-Tyr5]-bradykinin binding by PPCE were 5.1 +/- 2.3 X 10(-9) M, 1.2 +/- 0.3 X 10(-6) M and 1.4 +/- 0.6 X 10(-8) M.