A radioassay for aminoacylproline hydrolase (aminopeptidase P) activity

A radioassay was developed in which aminoacylproline hydrolase acts on Arg-Pro-Pro-[3H]benzylamide to yield arginine plus Pro-Pro-[3H]benzylamide. By stopping the reaction with base (0.1 M NaOH), the radioactive product is deprotonated to an organophilic form and is separable from the hydrophilic substrate by extraction of the alkaline aqueous solution with an organic solvent. When scintillants are included in the organic solvent, the enzyme:substrate reaction, extraction and quantification of Pro-Pro-[3H]benzylamide can all be conducted using a single liquid scintillation vial. Thus, aminoacylproline hydrolase activity is measured in terms of the rate of release of Pro-Pro-[3H]benzylamide. The substrate is obtainable at greater than 20 Ci/mmol, which enables its use under conditions of first-order enzyme kinetics. Conditions of near-zero order kinetics are readily attained by adding unlabeled substrate (Km 0.7 microM). The substrate is highly reactive (a 1:2000 dilution of guinea pig plasma hydrolyzed greater than 10% of the substrate during a 10 min incubation at 37 degrees C) and specific in that it is not degraded by leucine aminopeptidase, aminopeptidase A or N, dipeptidyl peptidase IV nor prolyl endopeptidase. The assay was used to measure aminoacylproline hydrolase specific activities in tissues of rat and guinea pig. Activity was found in virtually all major tissues of both species, and some guinea pig tissues (e.g. kidney and plasma) were found to be notably rich sources of the enzyme.