Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization

Abstract

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280 000 and 290 000 by nondenaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDSPAGE in the absence and presence of βME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km , Vmax , kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 M, 3.7 molper mg and min, 33.1 per s and 4.87 10,000 per s and M for AlaAlaPheMCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl 2 , and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide ArgValTyr from angiotensin III more rapidly than from AlaAlaPheMCA, and also released GlyAsnLeu from neuromedin B with the same velocity as from AlaAlaPheMCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as λRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > / = liver > heart > brain > lung > spleen >> skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.