BRASIL FACTOR - NEW PREKALLIKREIN ACTIVATOR in HUMAN-PLASMA

ESCOLA PAULISTA MED,DEPT BIOQUIM,São Paulo 01000,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,São Paulo 01000,SP,BRAZILWeb of Scienc

Characterization of a kinin inactivating serine endopeptidase H2 (kininase) from human urine using fluorogenic substrates

We have previously described a kinin-inactivating endopeptidase (H2), which was purified 19-fold from human urine by DEAE-cellulose chromatography and gel filtration. the enzyme was inhibited 100% by PMSF, TPCK and pOHMB. in the present communication, we further characterized this enzyme using the fluorogenic substrates Abz-RPPGFSPFRQ-EDDnp (Abz-BKQ-EDDnp) and Abz-FRQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). Also a rapid, sensitive and specific assay for the H2 was developed. the enzyme hydrolyzed bradykinin (BK = RPPGFSPFR) at the F-S peptide bond, differing from the cleavage site F-R, in the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FRQ-EDDnp. Other enzymes present. in urine als the serine endopeptidase III, prolyl endopeptidase and neutral endopeptidase-like were not able to hydrolyze the related substrate Abz-FRQ-EDDnp. the determined K-m for Abz-BKQ-EDDnp and Abz-FRQ-EDDnp were 0.79 mu M and 3.02 mu M, respectively. Using the fluorogenic substrates, we observed that PMSF and p-hydroxymercuribenzoate irreversibly inhibited the enzyme H2. E-64 was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. the inhibition observed in the presence of pOHMB was partially reversed by 2 mM cysteine. These results suggest that the H2 enzyme belongs to the subfamily of SH-containing serine proteases. Based on the molecular weight of isolated H2 (60 kDa), we believe that this enzyme originated from the kidney and may cleave the kinins filtered through the glomerulus and also that produced in the kidney. (C) 1999 Published by Elsevier Science B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Disciplina Nefrol, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Disciplina Nefrol, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023900 São Paulo, BrazilWeb of Scienc

HORSE URINARY KALLIKREIN .1. COMPLETE PURIFICATION and CHARACTERIZATION

ESCOLA PAULISTA MED,DEPT BIOQUIM,CP 20372,BR-04034 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOFIS,BR-04034 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,CP 20372,BR-04034 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOFIS,BR-04034 São Paulo,SP,BRAZILWeb of Scienc

TETRAPEPTIDE SUBSTRATES for the DISCRIMINATION AMONG KALLIKREINS and OTHER TRYPSIN-LIKE SERINE PROTEINASES

ESCOLA PAULISTA MED,DEPT BIOFIS,BR-01000 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT BIOFIS,BR-01000 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT BIOFIS,BR-01000 São Paulo,BRAZILWeb of Scienc

Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) activity was analyzed in human urine collected from mild hypertensive untreated patients. DEAE-cellulose chromatography using linear gradient elution revealed two forms of angiotensin I-converting enzyme, eluted in the conductivity of 0.75 and 1.25 mS. the fractions of each conductivity were pooled and submitted to direct eel filtration in an AcA-34 column, and the apparent molecular weights of urinary ACEs were estimated as 90 kDa (for ACE eluted in 0.75 mS) and 65 kDa (for ACE eluted in 1.25 mS). Both enzymes have a K-i of the order of 10(-7) M for the specific inhibitors studied, and are able to hydrolyze luteinizing hormone-releasing hormone and N-acetyl-Ser-Asp-Lys-Pro as described for N-domain ACE. By Western blot analysis, both peaks were recognized by ACE-specific antibody Y4, confirming the molecular weight already described. A plate precipitation assay using monoclonal antibodies to the N-domain of ACE showed that both forms of ACE binds with all monoclonal antibodies to the active N-domain ACE, suggesting that these forms of human urine ACEs resemble the N-fragment of ACE. the HP2 ACE (65 kDa) is similar to low molecular weight (LMW) ACE from normal subjects, and the HP2 ACE (90 kDa) is different from high molecular weight (190 kDa) and LMW (65 kDa) normal ACEs. the 90 kDa ACE could have an important role in development of hypertension. It will be fundamental to elucidate the molecular mechanism responsible for the genesis of this isoform. (C) 2001 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol, BR-04023900 São Paulo, BrazilFMUSP, HC, InCor, Dept Clin Med LIM13,Lab Genet & Cardiol Mol, BR-05403900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023900 São Paulo, BrazilWeb of Scienc

Purification and characterization of a neutral endopeptidase-like enzyme from human urine

Objective the aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP-like) in human urine end propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine.Methods Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FDQ-EDDnp. the K-m was determined using Abz-FDQ-EDDnp as a substrate. the hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). the mel. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast.Results the NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94 000. the substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. for this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined K-m was 14 mu M. the enzymatic activity was inhibited by thiorphan and phosphoramidon, in Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP.Conclusion A NEP-like enzyme was purified from human urine. Based on the mel. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. in the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP. J Hypertens 1998, 16:1971-1978 (C) 1998 Lippincott Williams & Wilkins.Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Nephrol Div,Disciplina Nefrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Med, Nephrol Div,Disciplina Nefrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, BrazilWeb of Scienc