Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates
- 作者：Quinto ; B.M.R. ; Juliano ; M.A. ; Hirata ; I. ; Carmona ; A.K. ; Juliano ; L. ; Casarini ; D.E.
- 关键词：Human urine ; Prolyl endopeptidase ; Kininase ; PE ; prolyl endopeptidase ; DEAE ; diethylaminethyl ; Abz ; ortho-aminobenzoic acid ; EDDnp ; N-[2 ; 4-dinitrophenyl]ethylenediamine ; TRH ; thyrotropin releasing hormone ; LHRH ; luteinising hormone releasing hormone ; BK
- 刊名：The International Journal of Biochemistry and Cell Biology
- 出版年：2000
- 期刊代码：127_13572725
- 类别：med
- 出版时间：December, 2000
- 卷：32
- 期：11-12
- 页码：1161-1172
- 文件大小：214 K
摘要
A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and it's analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100%by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for it's activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F–R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P–Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined Km for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was 
9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein–kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.
9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. It's known that all components from kallicrein–kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.