Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase a degrades collagen IV but not fibronectin

P. J. Kraft, D. E. Haynes-Johnson, L. Patel, J. A. Lenhart, R. A. Zivin, S. S. Palmer

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.

Original languageEnglish (US)
Pages (from-to)149-163
Number of pages15
JournalConnective Tissue Research
Volume42
Issue number2
DOIs
StatePublished - Jan 1 2001

Keywords

  • Collagen IV
  • Fibronectin
  • Fluorescence polarization
  • Gelatinase A
  • Matrilysin

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Nephrology
  • Cell Biology
  • Orthopedics and Sports Medicine

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