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 language | English |
---|---|
Pages (from-to) | 149-163 |
Number of pages | 15 |
Journal | Connective Tissue Research |
Volume | 42 |
Issue number | 2 |
State | Published - Nov 20 2001 |
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Keywords
- Collagen IV
- Fibronectin
- Fluorescence polarization
- Gelatinase A
- Matrilysin
ASJC Scopus subject areas
- Biochemistry
- Immunology
- Nephrology
- Cell Biology
- Orthopedics and Sports Medicine
Cite this
Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase a degrades collagen IV but not fibronectin. / Kraft, P. J.; Haynes-Johnson, D. E.; Patel, L.; Lenhart, J. A.; Zivin, R. A.; Palmer, S. S.
In: Connective Tissue Research, Vol. 42, No. 2, 20.11.2001, p. 149-163.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase a degrades collagen IV but not fibronectin
AU - Kraft, P. J.
AU - Haynes-Johnson, D. E.
AU - Patel, L.
AU - Lenhart, J. A.
AU - Zivin, R. A.
AU - Palmer, S. S.
PY - 2001/11/20
Y1 - 2001/11/20
N2 - 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.
AB - 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.
KW - Collagen IV
KW - Fibronectin
KW - Fluorescence polarization
KW - Gelatinase A
KW - Matrilysin
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UR - http://www.scopus.com/inward/citedby.url?scp=0034754758&partnerID=8YFLogxK
M3 - Article
C2 - 11718469
AN - SCOPUS:0034754758
VL - 42
SP - 149
EP - 163
JO - Connective Tissue Research
JF - Connective Tissue Research
SN - 0300-8207
IS - 2
ER -