Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization
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Pei-Ge Du
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.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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- Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization
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- Erratum
- Acknowledgement
- Content Index
- Author Index
- Subject Index
Articles in the same Issue
- New Control of Mitochondrial Membrane Potential and ROS Formation A Hypothesis
- Brix from Xenopus laevis and Brx1p From Yeast Define a New Family of Proteins Involved in the Biogenesis of Large Ribosomal Subunits
- Mig-6 Is a Negative Regulator of the Epidermal Growth Factor Receptor Signal
- β-Carotene Inhibits Growth of Human Colon Carcinoma Cells in Vitro by Induction of Apoptosis
- Ligand-Mediated Protection against Phage Lysis as a Positive Selection Strategy for the Enrichment of Epitopes Displayed on the Surface of E. coli Cells
- Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm
- Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase
- Total Antioxidant Capacity and Nuclear DNA Damage in Keratinocytes after Exposure to H2 O2
- Recombinant Cryptic Human Fibronectinase Cleaves Actin and Myosin: Substrate Specificity and Possible Role in Muscular Dystrophy
- Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization
- Determination of NADH in Frozen Rat Brain Sections by Laser-Induced Fluorescence
- Human -Calpain: Simple Isolation from Erythrocytes and Characterization of Autolysis Fragments
- Erratum
- Acknowledgement
- Content Index
- Author Index
- Subject Index
