A specific tripeptidyl substrate for tripeptidyl peptidase activity is effectively hydrolyzed by alanyl aminopeptidase/aminopeptidase N/CD13 in the rat kidney

L-Alanyl-L-alanyl-L-phenylalanine 4-methylcoumaryl-7-amide (AAF-MCA) is one of the classic substrates for use with tripeptidyl peptidases (TPP-I and TPP-II). We have previously clarified the tissue distribution of TPP-I in detail and noted that the protein expression of TPP-I is often incompatible with its enzyme activity. Herein, we describe the unknown peptidase, which could effectively hydrolyze AAF-MCA, in the rat kidney. The peptidase was purified after four chromatography steps, and its enzyme characteristics were elucidated. The peptidase activity was inhibited by amastatin, bestatin, and o-phenanthroline and was also inhibited by zinc and copper ions. The substrate specificity for several monoamino acidic-MCAs revealed that the peptidase had an affinity for alanyl-MCA. The amino terminal amino acid sequence of the peptidase was x-Ala-Pro-x-Leu-Pro-Gly-Ser-Thr-Ser-Ala-Thr-x-x-Ser, where x indicates undetectable amino acid residues, and the antiserum against the peptidase was immunopositive for the brush border of a renal proximal tubule and the small intestine, and the surface membrane of bile canaliculi. These results indicate that the unknown peptidase that hydrolyzed AAF-MCA is the soluble form of aminopeptidase N/CD13, and caution is required when using AAF-MCA as a substrate for tripeptidyl peptidase assays.