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 and 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 Km 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 mol. 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 94000. 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 Km was 14 microM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP.


CONCLUSIONS
A NEP-like enzyme was purified from human urine. Based on the mol. 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.
Di Marco
G S
GS
Department of Medicine, EPM, UNIFESP, São Paulo, SP, Brazil.
Quinto
B M
BM
Juliano
M
M
Carmona
A K
AK
Stella
R C
RC
Plavnik
F L
FL
Casarini
D E
DE
D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000652:urine*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D015260:Neprilysin