Purification of This Endopeptidase by Affinity Chromatography*

The endopeptidase, post-proline cleaving enzyme, has been purified 10,500-fold in an overall yield of 18% from lamb kidney. The enzyme possesses a specific activity of 45 ~mol/mg/min as tested with the substrate Z-Gly-Pro-Leu-Gly (K,,, = 6.0 x 10m5), has a molecular weight of 115,000, is comprised of two subunits with a molecular weight of 57,000, and exhibits maximal activity at pH 7.5 to 8.0. With the exception of the -Pro-Prolinkage, the -Pro-X-peptide bond (X equals Land n-amino acid residues) located internally in the peptide sequence can be hydrolyzed (cleavage occurs faster when X = lipophilic side chain as compared to X = acidic side chain). The appropriate -Pro-Xbonds in zinc-free porcine insulin, oxytocin, arginine vasopressin, angiotensin II, bradykinin-potentiating factor were cleaved. Human gastrin, adrenocorticotropic hormone, denatured guinea pig skin collagen, and ascaris cuticle collagen were not degraded. Dipeptides with the structure Z-Pro-m-X competitively inhibit post-proline cleaving enzyme.