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 1:1/3 ratio) in the processing of the human Ang-(1-12) (hAng-(1-12)) substrate. To determine the K-m and V-max , the CPA and recombinant human chymase (rhChymase) enzymes were incubated with increasing concentrations of hAng-(1-12) substrate (0-300 mu M). 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, I-125-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 1:1/3 ratio), the amount of Ang II formation from I-125-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 I-125-hAng-(1-12). The K-m and V-max values were 150 +/- 5 mu M and 384 +/- 23 nM/min/mg of CPA and 40 +/- 9 mu M and 116 +/- 20 nM/min/mg of rhChymase. The catalytic efficiency (V-max /K-m 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. (C) 2019 Elsevier Inc. All rights reserved.