PMID- 8660561
OWN - NLM
STAT- MEDLINE
DCOM- 19960925
LR  - 20061115
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 237
IP  - 2
DP  - 1996 Jun 1
TI  - A highly selective assay for neutral endopeptidase based on the cleavage of a 
      fluorogenic substrate related to Leu-enkephalin.
PG  - 167-73
AB  - An intramolecularly quenched fluorogenic peptide structurally related to 
      Leu-enkephalin, containing o-aminobenzoyl (Abz) and ethylenediamine 
      2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid 
      residues, Abz-Gly-Gly-D-Phe-Leu-Arg-Arg-Val-EDDnp (Abz-GGDFLRRV-EDDnp), was 
      selectively hydrolyzed at the Arg-Val bond by neutral endopeptidase (NEP, 
      enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, 
      kcat = 127 min-1 and kcatsolidusKm = 42 min-1 microM-1) similar to those of the 
      Leu-enkephalin. The specificity of the NEP assay was demonstrated by incubating 
      Abz-GGDFLRRV-EDDnp with a kidney homogenate or with crude membrane preparations 
      of brain and lung: more than 95% of all products released were the complementary 
      fragments Abz-GGDFLRR and V-EDDnp which were totally inhibited by 1 microM 
      thiorphan, a highly specific NEP inhibitor. The blocked amino- and 
      carboxyl-terminal amino acids protected this substrate against the action of 
      aminopeptidases as well as of carboxypeptidases. Furthermore, D-Phe amino acid 
      also ensured a very good protection of Abz-GGDFLRRV-EDDnp against the action of 
      other tissue endopeptidases distinct from NEP. A continuous fluorometric assay 
      for only 5 min was sufficient to quantify the NEP activity with a minimum 
      sensitivity of 5 ng of purified enzyme or the equivalent enzymatic activity in 
      crude tissue preparations. Therefore, amounts as little as 0.5 ng of enzyme could 
      be quantified employing longer times of incubation.
FAU - Carvalho, K M
AU  - Carvalho KM
AD  - Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara, 
      Fortaleza, 60430-270, Brazil.
FAU - Boileau, G
AU  - Boileau G
FAU - Camargo, A C
AU  - Camargo AC
FAU - Juliano, L
AU  - Juliano L
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Recombinant Proteins)
RN  - 58822-25-6 (Enkephalin, Leucine)
RN  - EC 3.4.24.11 (Neprilysin)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Enkephalin, Leucine/analogs & derivatives/chemistry
MH  - Fluorescent Dyes/chemistry
MH  - Humans
MH  - Hydrolysis
MH  - Kinetics
MH  - Molecular Sequence Data
MH  - Neprilysin/*analysis/*metabolism
MH  - Recombinant Proteins/analysis/metabolism
MH  - Sensitivity and Specificity
MH  - Spectrometry, Fluorescence/statistics & numerical data
MH  - Substrate Specificity
EDAT- 1996/06/01 00:00
MHDA- 1996/06/01 00:01
CRDT- 1996/06/01 00:00
PHST- 1996/06/01 00:00 [pubmed]
PHST- 1996/06/01 00:01 [medline]
PHST- 1996/06/01 00:00 [entrez]
AID - S0003-2697(96)90224-9 [pii]
AID - 10.1006/abio.1996.0224 [doi]
PST - ppublish
SO  - Anal Biochem. 1996 Jun 1;237(2):167-73. doi: 10.1006/abio.1996.0224.