Metabolism of vasoactive peptides by plasma and purified renal aminopeptidase M

Aminopeptidase M (AmM; EC 3.4.11.2) is a membrane-bound peptidase present on renal brush border and vascular plasma membrane. In the present study, AmM, purified from rabbit kidney cortex, produced a single immunoprecipitin line against AmM antisera, hydrolyzed alanyl-, leucyi- and arginyl-β-naphthylamides at rates of 5.1 ± 0.5, 3.9 ± 0.5 and 2.6 ± 0.3 μmol/min/mg, respectively, exhibited little or no α-glutamyl-, aspartyl- or glycyl-prolyl-naphthylamidase activities (≤0.14 μmol/ min/mg), and was inhibited by o-phenanthroline, amastatin (ic₅₀ = 400 nM) and bestatin (ic50 = 6 μM). The alanyl-naphthylamidase activity of unfractionated rabbit plasma was found to be identical to purified AmM regarding relative rates of hydrolysis of alanyl-, leucyl- and arginyl-naphthylamides (100:79:42), pH Optimum, and inhibition profile. In comparative studies with the purified enzyme, immunoreactive AmM accounted for essentially all of the alanyl-2-naphthylamidase activity of rabbit plasma. N-Terminal metabolism of (Met⁵)enkephalin by purified renal AmM was 3.92 ± 0.69 μmol/min/mg, followed by somatostatin (1.25 μmol/min/mg), hepta(5-11)substance P (1.14 ± 0.13 μmol/min/mg), (Asn¹)angiotensin II (1.11 ± 0.06 μmol/min/mg), angiotensin III (0.45 ± 0.04 μmol/min/mg) and des(Asp¹)-angiotensin I (0.36 ± 0.04 μmol/min/mg). In contrast, substance P, bradykinin, (Sar¹,Ala⁸)angiotensin II and neurokinin analogs containing modified N-termini (e.g. Ac-Arg) were resistant to hydrolysis by AmM. Peptide degradation was optimal at neutral pH and was inhibited by amastatin (ic50 = 200 nM) and bestatin (ic50= 5 μM). Apparent K_m values ranged from 15.7 ± 0.4 μM for angiotensin III to 102 ± 2 μM for (Met⁵)enkephalin. These data support a significant role for vascular and plasma AmM in the metabolism of circulating vasoactive peptides.