Mast cell peptidases (carboxypeptidase A and chymase)-mediated hydrolysis of human angiotensin-(1–12) substrate

Abstract

Angiotensin processing peptidases (carboxypeptidase A (CPA) and chymase) are stored in cardiac mast cell (MC) secretory granules in large quantity and are co-released into the extracellular environment after activation/degranulation. In the human heart, chymase is primarily responsible for angiotensin II (Ang II) generation from the alternate substrate angiotensin-(1–12) (Ang-(1–12)). We investigated the individual and combined hydrolytic specificity of CPA and chymase enzymes (1:1 and 1:⅓ ratio) in the processing of the human Ang-(1–12) (hAng-(1–12)) substrate. To determine the Km and Vmax, the CPA and recombinant human chymase (rhChymase) enzymes were incubated with increasing concentrations of hAng-(1–12) substrate (0–300 μM). We found that CPA alone sequentially metabolized hAng-(1–12) substrate into angiotensin-(1–9) (Ang-(1–9), 53%), Ang II (22%) and angiotensin-(1–7) (Ang-(1–7), 11%) during a 15 min incubation. In the presence of rhChymase alone, 125I-hAng-(1–12) was directly metabolized into Ang II (89%) and no further hydrolysis of Ang II was detected. In the presence of both CPA + rhChymase enzymes (1:1 or 1:⅓ ratio), the amount of Ang II formation from 125I-hAng-(1–12) within a 5 min incubation period were 68% or 65%, respectively. In the presence of both (CPA + rhChymase), small amounts of Ang-(1–9) and Ang-(1–7) were generated from 125I-hAng-(1–12). The Km and Vmax values were 150 ± 5 μM and 384 ± 23 nM/min/mg of CPA and 40 ± 9 μM and 116 ± 20 nM/min/mg of rhChymase. The catalytic efficiency (Vmax/Km ratio) was higher for rhChymase/hAng-(1–12) compared to CPA/hAng-(1–12). Compared to CPA, chymase has a much higher affinity to hydrolyze the hAng-(1–12) substrate directly into Ang II. In addition, Ang II and Ang-(1–7) are the end products of chymase and CPA, respectively. Overall, our findings suggest that the Ang II generation from hAng-(1–12) is primarily mediated by chymase rather than CPA.