Human skin chymotrypsin-like proteinase, human neutrophil cathepsin G, rat mast cell chymase, and rat salivary gland tonin are cell-derived serine proteinases of similar size with specificity for amino acids of aromatic residues. Each enzyme was examined for its ability to convert angiotension I to angiotensin II and to cleave a panel of synthetic substrates. Skin chymotryptic proteinase, cathepsin G, and tonin cleaved the phe8-his9 bond of angiotensin I and converted angiotensin I to angiotensin II without further degradation. In contrast, chymase formed relatively small amounts of angiotensin II because it preferentially cleaved the tyr4-ile5 bond of angiotensin I. The rank order of angiotensin I converting activity was skin chymotryptic proteinase greater than tonin greater than cathepsin G greater than chymase. The Km and Kcat for angiotensin I conversion by the human skin enzyme were 6.6 X 10(-5) M and 50 s-1, respectively. The angiotensin I converting activity of human skin chymotryptic proteinase is equal to or greater than the peptidyl dicarboxypeptidase angiotensin-converting enzyme. Substrate specificities of each enzyme were further distinguished by use of benzoyl-L-tyrosine ethyl ester. A limited immunologic characterization of each enzyme was performed with monospecific goat antiserum to cathepsin G and chymase by Ochterlony gel diffusion. Each antiserum gave a precipitin line against its respective immunogen without evidence of cross-reactivity against the other enzymes. Human skin chymotryptic proteinase, cathepsin G, and tonin provide unique pathways for the generation of angiotensin II in tissue and may be of significance in regulation of biologic processes of the tissue microenvironment. The kinetic constants of the human skin chymotryptic proteinase for angiotensin I conversion, are consistent with the potential to carry out a reaction of physiologic importance.

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Proteases from human neutrophils can generate angiotensin II directly from angiotensin I or angiotensinogen. We examined whether neutrophil protease also influences angiotensin formation by activating human prorenin (also called inactive renin). When incubated with partially purified plasma and amniotic prorenin, sonicates from 10 6 neutrophils resulted in 120 ± 30% and 1,240 ± 290% increase in renin activity, respectively. The pH optimum of neutrophil prorenin-activating enzyme(s) is 6.5-7.0, and the activity of the enzyme(s) is inhibited by a mixture of serine protease inhibitors but not by inhibitors of other proteases, suggesting that prorenin-activating enzyme(s) is a neutral serine pro-tease^). Stimulation of neutrophils by f-met-leu-phe in the presence of cytochalasin B resulted in release of prorenin-activating enzyme(s) in a dose-dependent fashion. We attempted to isolate prorenin-activating enzyme(s) from neutrophil granules using aprotinin-affinity and carboxymethyl cellulose chromatographies. Prorenin-activating enzyme(s) coeluted with cathepsin G and elastase activities. Prorenin activation was greatly inhibited by anticathepsin G antiserum. Purified cathepsin G activated prorenin in a dose-dependent fashion. Elastase probably also contributes to prorenin activation since purified elastase also activated human prorenin. We speculate that this neutrophilic angiotensin-generating system may play a role in the local generation and concentration of angiotensins by influencing multiple steps of the renin-angiotensin system. (Circulation Research 1987;60:595-601)

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his, bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by i0' M phenylmethylsulfonylfluoride but not by 1O-3 M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsinlike proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10-5 M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.