Putative tripeptidyl peptidase in renal brush border

To the Editor: We wish to draw readers’ attention to the need for caution in interpreting assays of peptidases using fluorogenic substrates such as Gly-Pro-Met-2 naphthylamide (NNap). The release of the fluorophore, 2naphthylamine, may indicate the action of a tripeptidyl peptidase releasing the tripeptide, Gly-Pro-Met, but it may also result from the sequential attack of other exopeptidases, first releasing Gly-Pro and then free methionine. The essential criterion of a tripeptidyl peptidase attack is the demonstration of the tripeptide among the products of the reaction. Two recent reports by Knut-Jan Andersen and J. Ken McDonald (1, 2) of a tripeptidyl peptidase active at pH 7 in kidney brush-border membranes do not contain any evidence that a tripeptide was released from the naphthylamide substrate. We have now reinvestigated this question and fail to find a tripeptidyl peptidase hydrolyzing either Gly-Pro-Met-NNap or Gly-Pro-Leu-NNap in pig or rat microvillar membranes (3). The hydrolysis of both substrates was readily assayed fluorimetrically, but the products analyzed by high precision liquid chromatography were Gly-Pro, Met-NNap (or Leu-NNap) and free naphthylamine, with no evidence of tripeptidases. Moreover, the activity monitored by the assay was inhibited by either diisopropylfluorophosphate or amastatin (inhibitors of dipeptidyl peptidase IV and aminopeptidase N, respectively) and could be titrated to zero by polyclonal antibodies raised to pig and rat aminopeptidase N. It was our conclusion that these substrates were hydrolyzed by renal microvilli in a two-step process, first the release of Gly-Pro by dipeptidyl peptidase IV and then the hydrolysis of the amino acid naphthylamide by aminopeptidase N. There are twelve well-characterized peptidases so far identified in renal microvilli (4), but a true tripeptidyl peptidase cannot yet be added to the list.