An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

Anal Biochem. 2016 Aug 15:507:13-7. doi: 10.1016/j.ab.2016.05.001. Epub 2016 May 10.

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.

Keywords: ADAM10; ADAM17; ADAM9; Assay; Extracellular vesicles; Fluorescent substrate.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ADAM Proteins / analysis*
  • ADAM Proteins / chemistry
  • ADAM Proteins / metabolism*
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemistry*
  • Humans
  • Hydrolysis
  • Regression Analysis
  • Solubility

Substances

  • Fluorescent Dyes
  • ADAM Proteins