Neumann, Ulf, Kubota, Hisashi, Frei, Karl, Ganu, Vishwas and Leppert, David (2004) Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme. Analytical Biochemistry, 328 (2). pp. 166-173. ISSN 0003-2697

Abstract

Matrix metalloproteinases (MMPs) and the related tumor necrosis factor converting enzyme (TACE) are involved in tissue remodeling, cell migration, and processing of signaling molecules, such as cytokines and adhesion molecules. Fluorescence-quenched peptide substrates have been widely used to quantitate the actual enzymatic activity of MMPs. However, the various MMPs have very different specific activities toward these substrates. This restricts their value for the determination of composite proteolytic activity of mixtures of metalloproteinases in biological fluids. The N-terminal elongation of the most widely used MMP substrate (FS-1) with a Lys to the sequence Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2) (FS-6) yields a fluorogenic peptide with improved substrate properties. As compared to FS-1, the specificity constant (kcat/Km) of FS-6 for collagenases (MMP-1, MMP-8, MMP-13) and MT1-MMP (MMP-14) is increased two- to ninefold and threefold, respectively, while those for gelatinases and matrilysin remain equally high. Using high-performance liquid chromatography-fluorescence detection, MMP activity can be quantitated in the picomolar range. FS-6 shows up to twofold higher specificity constants (kcat/Km of 0.8x10(6)M(-1)s(-1)) for TACE, as compared to standard substrates Mca-PLAQAV-Dpa-RSSSAR-NH(2) and Dabcyl-LAQAVRSSSAR-EDANS. FS-6 is fully water soluble and thus allows measurement of metalloproteinase activity in tissue culture conditions, e.g., on the surface of viable cells in situ.

Item Type:	Article
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2003.12.035
Additional Information:	author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used
Keywords:	Fluorogenic enzyme substrate; MMP; TACE; Kinetics
Related URLs:	http://www.ncbi.nlm.nih.gov/sites/entrez... doi:10.1016/j.ab.2003.12.035
Date Deposited:	14 Dec 2009 14:01
Last Modified:	31 Jan 2013 01:20
URI:	https://oak.novartis.com/id/eprint/319