Histochemical demonstration of tripeptidyl aminopeptidase I in rat organs

Tripeptidyl aminopeptidase I (TPPI) is presently a subject of substantial research interest due to its wide tissue distribution and multiple functions in mammalians, including humans. TPPI is a lysosomal enzyme belonging to the S53 sedolisin family of serine-carboxyl peptidases. It hydrolyzes tripeptides from the N-terminal of oligo- and polypeptides at an optimum of pH 4.5. At lower pH values (3.0-3.5) it possesses endopeptidase activity. The enzyme is a highly conservative glycoprotein, widely distributed in all the mammalian species. The aim of the present study was to demonstrate TPPI activity in various tissues and organs in Wistar rats by means of recently developed enzyme histochemical methods. The detection procedures are based on two specific substrates for TPPI, glycyl-L-prolyl-L-metionyl-5-chloro-1-anthraquinonylhydrazide (GPM-CAH) and glycyl-L-prolyl-L-metionyl-4-hydrazido-N-hexyl-1,8-naphthalimide (GPM-HHNI), for chromogenic and fluorogenic histochemical demonstration of the enzyme, respectively. In this study, the enzyme reaction was seen in the cerebral and cerebellar cortex, in the hepatocytes in the liver, in the bronchial epithelial cells and alveolar macrophages in the lung, in the epithelial and interstitial cells of the epididymis and in the glomus and sustentacular cells of the carotid body. Using the chromogenic substrate, the enzyme locations were visible as dark orange to brown granules around the cell nuclei. In the cells with a low enzyme activity, the final reaction product was observed as an almost homogenous yellowish precipitate. Using the fluorogenic substrate, the final enzyme reaction product could be observed both in brightfield and fluorescence fields. In the brightfield images it appeared as orange-red to brownish granules around the cell nuclei. In fluorescent images, the reaction product was clearly visible as orange-red to bright red granules. Our results show that TPPI is widely distributed in rat tissues and organs. The present findings are largely consistent with those obtained by immunohistochemical methods for visualization of the enzyme