A selective assay for endooligopeptidase A based on the cleavage of fluorogenic substrate structurally related to enkephalin.
A novel quenched fluorescence substrate, QF-ERP7 (Abz-G-G-F-L-R-R-V-EDDn), structurally related to enkephalins, proved to be suitable for assaying the endooligopeptidase A (E.C.3.4.22.19) activity. The enzyme only splits the L-R bond (Km 1.75 microM, Kcat 8.25 s-1), a reaction efficiently blocked by anti-endooligopeptidase A antibodies and by inhibitor and alternative substrates of the enzyme. Evidences based on the action of inhibitors and on the analysis of QF-ERP7 fragments demonstrated that endooligopeptidase A contributes with 100% of the QF-ERP7 cleaving activity found in the cytosol of rabbit brain homogenates and with 85% of that recovered in the membrane fraction. Homologous substrates, Abz-G-G-F-L-R-R-EDDn and Abz-G-G-F-L-R-EDDn, were resistant to hydrolysis. The convenience and sensitivity of the fluorimetric assay based on the QF-ERP7 moiety offers several advantages compared with previously described painstaking procedures for endooligopeptidase A activity measurements, what will certainly contribute to further our understanding of the role of this enzyme on the peptide hormone metabolism.
Juliano
L
L
Department of Biophysics, Escola Paulista de Medicina, São Paulo, SP, Brazil.
Chagas
J R
JR
Hirata
I Y
IY
Carmona
E
E
Sucupira
M
M
Oliveira
E S
ES
Oliveira
E B
EB
Camargo
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000276:immunology; D004745:Enkephalins; D005029:Ethylenediamines; D005453:Fluorescence; D006868:Hydrolysis; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011817:Rabbits
D000906:Antibodies; D004745:Enkephalins; D005029:Ethylenediamines; D009842:Oligopeptides; C066641:QF-ERP7; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase