Bmr. Quinto et al., Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates, INT J BIO C, 32(11-12), 2000, pp. 1161-1172
Citations number
34
Categorie Soggetti
Biochemistry & Biophysics
Journal title
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAF-
cellulose and Sepharose Mercurial chromatographies. In this work we studied
the specificity of PE using different fluorogenics substrates. Further cha
racterization 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 p
rolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophe
nyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specifi
c for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhi
bited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phos
phoramidon, when we used the mentioned substrates. These results suggest th
at 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 substr
ate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urin
ary PE is particularly unable to hydrolyze peptides with single prolines su
ch as substance P, neurotensin and LHRH. The determined K-m for Abz-RPPGFSP
FRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimu
m pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was simi
lar to 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 t
ubules, have an important role in kidney handling of many peptides, which a
re filtered by the glomerulus. The prolyl endopeptidase located at distal t
ubule could have an important physiological function in control of kinin fo
rmed in this portion. It's known that all components from kallicrein-kinin
system like low molecular weigh kininogen and kallikrein are presents in th
is portion. (C) 2000 Elsevier Science Ltd. All rights reserved.