Substrate Specificity of Aminopeptidase M: Evidence that the Commercial Preparation Is Contaminated by Dipeptidyl Aminopeptidase IV and Prolidase

Abstract

Commercial preparations of aminopeptidases M split Gly-Pro-β-naphthylamide (Gly-Pro-2-NNap) into Gly-Pro and β-naphthylamine, and Ala-Pro into Ala and Pro. The activities on Gly-Pro-2-NNap and Ala-Pro were completely inhibited by di-isopropyl phosphoro fluoridate (DFP) and p-chloromercunbenzoate (PCMB), respectively. When the substrate specificity was analyzed with tuftsin, Thr and Lys-Pro-Arg were released, and then Lys-Pro-Arg was split into Lys-Pro and Arg. Thereafter, slow liberation of Lys and Pro from Lys-Pro took place. The DFP-treated enzyme released only Thr from tuftsin and no hydrolysis of Lys-Pro-Arg was observed. With the enzyme treated with PCMB, tuftsin was converted into Thr and Lys-Pro-Arg, followed by the liberation of Arg, but no release of Lys and Pro was observed, contrary to the case of the untreated-enzyme. These results show that commercial aminopeptidase M contains dipeptidyl aminopeptidase IV and prolidase. Contamination by dipeptidyl aminopeptidase IV was confirmed by an immunological method.