New Fluorogenic Substrates for N-Arginine Dibasic Convertase Eva Csuhai,* Maria Aparecida Juliano,† Jan St. Pyrek,‡ Amy C. Harms,‡ Luiz Juliano,† and Louis B. Hersh* *Department of Biochemistry and ‡Mass Spectrometry Facility, University of Kentucky, Lexington, Kentucky, 40536-0084; and †Department of Biophysics, Escola Paulista de Medicina, Sa ˜ o Paulo, Brazil Received October 16, 1998 N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The charac- terization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLR- RIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2,4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectros- copy. The rate of hydrolysis of the fluorogenic sub- strates is proportional to enzyme concentration, and obeys Michaelis–Menten kinetics. The kinetic param- eters for the fluorescent peptides (K m values of 1.0 M, and V max values of 1 M/(min  mg) are similar to those obtained with peptide hormones as substrates. © 1999 Academic Press Peptidases have been found to play an important role in modulating the action of peptide hormones. Peptidases can hydrolytically inactivate a peptide, can convert a peptide hormone precursor to an active form, or can alter the physiological activity of the parent peptide. Recently, a novel endopeptidase, N-arginine dibasic convertase (NRD convertase), 1 was described (1, 2). NRD convertase is a 130-kDa metallopeptidase which cleaves peptide bonds between paired basic se- quences of the type Arg-Arg or Arg-Lys (1, 3). The enzyme is broadly expressed in tissues, being most abundant in testes, with high enzyme activity, as well as mRNA, also found in brain and spinal cord (4, 5). The enzyme appears to be developmentally regulated; at early stages of embryonic development it is detected exclusively in brain and spinal cord. At later develop- mental stages this peptidase appears to be expressed ubiquitously (6). It is interesting to note that NRD convertase is abundant in all immortalized cell lines examined to date (5, 7). NRD convertase is a soluble protein found in the cytosol as well as in secretory vesicles (8). In addition, a secreted form has been reported (3). This endopepti- dase is a member of a relatively new family of metal- lopeptidases, the Zn-containing inverzincins. Other members of this group include insulin-degrading en- zyme and protease III of Escherichia coli. The putative active site in this peptidase family is characterized by an inverted Zn-binding motif HXXEH (9). The mecha- nism of peptide cleavage at this inverted Zn-binding site has not yet been fully characterized. NRD conver- tase has been shown to cleave a relatively narrow range of substrates in vitro, including the opioid pep- tides dynorphin A and B, bovine adrenal medulla pep- tide, and -neoendorphin (3). The in vivo substrate specificity of the enzyme, however, has yet to be deter- mined. A distinguishing characteristic of NRD convertase is an extensive acidic domain consisting of 43–59 nega- tively charged residues within a 76-amino-acid stretch 1 Abbreviations used: NRD convertase, N-arginine dibasic conver- tase; Abz, 2-aminobenzoyl; EDDnp, ethylenediamine-2,4-dinitrophe- nyl; TBTU, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tet- rafluoroborate; Hobt, 1-hydroxybenzotriazole; NMM, N-methyl morpholine; MALDI–TOF, matrix-assisted laser desorption ioniza- tion–time of flight; Fmoc, 9-fluorenylmethoxycarbonyl. 0003-2697/99 $30.00 149 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 269, 149 –154 (1999) Article ID abio.1999.4033, available online at http://www.idealibrary.com on