DI- and Tripeptidyl Carboxypeptidase and Endopeptidase Activities of Angiotensin Converting Enzyme from Rabbit Brain and Lung
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Angiotensin converting enzyme is a dipeptidyl carboxypeptidase, cleaving dipeptides from the unblocked C-terminus of peptide substrates without a penultimate proline residue. Recently, novel activities for this enzyme have been reported, including the production of tripeptides from substrates with penultimate proline, and endopeptidase activity using substrates with a blocked C-terminus, including the neuropeptide substance P. These novel activities were investigated. using enzyme from rabbit brain and lung that was purified by a new method using a single affinity column, which provided yields of exceptional quantity and purity. Comparison of the physicochemical properties and dipeptidyl carboxypeptidase activities of the two enzyme preparations indicated they were identical. Both enzymes exhibited tripeptidyl carboxypeptidase activity with the substrates des-Arg9-bradykinin and Fa-Phe-Ala-Pro-Phe. Kinetic parameters for the latter substrate were obtained using a continuous spectrophotometric assay method. Both preparations also showed endopeptidase activity with substance P, and the artificial substrates Abz-AAYLAG-Nbz and Fa-Phe-Ala-Pro-Phe methyl ester, with kinetic parameters for the latter again obtained by spectrophotometry. Modification of arginine in the active site of the lung enzyme indicated this residue was critical to all three activities. These studies confirm ~ that novel activities are an intrinsic property of angiotensin converting enzyme, independent of species, tissue or substrate, and show that simple assay methods using Fa-peptide substrates may be adapted to their investigation. Although the enzyme hydrolyzes substance P, it is unlikely that this is a physiologic mechanism, and the possible physiologic roles of these novel activities remain unknown.