Evaluation of seprase activity.
Seprase is a serine protease that is integral to the plasma membrane and is overexpressed by invasive tumor cells (Piñeiro-Sánchez et al., J Biol Chem 1997; 272: 7595-601; Monsky et al., Cancer Res 1994; 54: 5702-10). Seprase activity is most often assessed by zymography, which is not a quantitative assay. This study establishes a relatively simple and quantitative method for determining seprase activity. The degradation of a 3H-gelatin substrate is measured in the presence of 5 mM EDTA which inhibits matrix metalloproteinases but not seprase. The quantitative character of the assay was demonstrated using partially purified seprase from chicken embryos, a preparation that lacks detectable matrix metalloproteinase activity. In this assay, release of 3H-gelatin fragments is linear over time for 1.5 microg/assay seprase concentration as well as for preparations concentrated or diluted by five fold (7.5 microg/assay and 0.3 microg/assay respectively). Additional experiments were performed to validate the quantification of seprase activity using the radiographic assay by comparing the results to zymography. Exposure to 22 or 37 degrees C results in maximal seprase activity while exposure to 80 or 100 degrees C completely abolishes seprase activity in both zymography and the radiographic assay. Exposure to 60 degrees C abolished seprase activity as judged by zymography, but about 50% gelatinase activity was observed using the 3H-gelatin substrate. Immunopreciptiation with seprase-specific antibody specifically removed seprase and lowered the seprase activity remaining in the extracts as judged by both assays. Investigation of the seprase that was partially purified from human breast cancer tissue revealed that its specific activity (cpm gelatin fragments released/{mg protein x h}) is five times greater than that of seprase purified from chicken embryos. This assay will be useful for determining the seprase activity in extracts of tumor tissues and cells as well as for identifying inhibitors of seprase.
Kelly
T
T
Department of Pathology, Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock 72205-7199, USA. kellythomasj@exchange.uams.edu
D000818:Animals; D014408:Biomarkers, Tumor; D002642:Chick Embryo; D010450:Endopeptidases; D005069:Evaluation Studies as Topic; D018093:Gelatinases / Q000378:metabolism*; D016014:Linear Models; D008565:Membrane Proteins; D051381:Rats; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha
D014408:Biomarkers, Tumor; D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D000099167:Fibroblast Activation Protein Alpha