title: An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities.
abstract: A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.
authors.affiliation: BioZyme, Apex, NC 27523, USA. Electronic address: moss0610@yahoo.com.
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Moss
authors.affiliation: Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
authors.affiliation: Department of Pathology, University of Pittsburgh Cancer Institute, VA Healthcare System, Pittsburgh, PA 15232, USA.
authors.forename: Toshie
authors.initials: T
authors.lastname: Yoneyama
authors.affiliation: Department of Internal Medicine I, University Clinic Cologne, Cologne 50937, Germany.
authors.forename: Hinrich P
authors.initials: HP
authors.lastname: Hansen
authors.affiliation: Department of Pathology, University of Pittsburgh Cancer Institute, VA Healthcare System, Pittsburgh, PA 15232, USA.
authors.forename: Nikola
authors.initials: N
authors.lastname: Vujanovic
authors.affiliation: BioZyme, Apex, NC 27523, USA.
authors.forename: Fred H
authors.initials: FH
authors.lastname: Rasmussen
mesh_terms: D051722:ADAM Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D012044:Regression Analysis; D012995:Solubility
chemical_list: D005456:Fluorescent Dyes; D051722:ADAM Proteins
keywords: ADAM10; ADAM17; ADAM9; Assay; Extracellular vesicles; Fluorescent substrate