Studies Of Rat Tripeptidyl Peptidase Ⅰ On The Gene Expression,Purification And Hydrolysis To Angiotensin

Tripeptidyl peptidase I (TPP I) was purified to homogeneity from a rat kidney lysosomal fraction. The molecular weight of the enzyme was calculated to be 280KD on non-denaturing PAGE and gel filtration, respectively, and to be 43KD and 46KD on SDS-PAGE in the absence and presence of β-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The enzyme rapidly hydrolyzed the substrate Ala-Ala-Phe-MCA in a pH range of 3.5 to 4.5, and it was stable from pH 2 to 7 and up to 50(C. The Km and Vmaxvalues of TPP I at optimal pH 4.0 were 680 μM, 3.7μmol..min-1for Ala-Ala-Phe-MCA, respectively. TPP I was potently inhibited by DFP and HgCl2, and moderately by PCMBS. These findings also suggest that TPP I is an exo-type serine peptidase that is regulated by SH reagent. For further information on the molecular structure, we screened a rat liver cDNA library using a DNA probe that originated from human TPP I,and we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA designated as RTI-1 is composed of 2,485 bp of nucleotides in length and encodes 563 amino acids in the coding region. In Northern blot analysis, the order for TPP I mRNA expression was kidney ≥ liver > heart > brain > lung > spleen >> skeletal muscle and testis. In parallel with northern blot analysis, the TPP I antigen was detected in various rat tissues by immunohistochemical staining. To investigate the bio-activity of TPP I from rat kidneys, especially the hydrolysis mechanism of small molecular peptides, effects of digestion of angiotensins I.II.III and other small molecular peptides were analyzed by HPLC and TOF-MS. The data suggest that N-termini of angiotensins I.II.III were cleaved of different degrees tripeptides with purified TPP I .And TPP I released the tripeptide Arg-Val-Try from angitensin III more rapidly than from Ala-Ala-Phe-MCA. Namely, the relatiove order of cleavage by TPP I was : angiotensin III >> Ala-Ala-Phe-MCA >angiotensi II> angiotensi I。 Accordingly , It is thought that angiotensin III is good natural substrates for TPP I .