Cell associated elastase activities of rat mammary tumour cells.
As part of our studies into the role of tumour cell proteinases in cancer invasion, we have adapted a fluorogenic assay to measure the elastase activities of intact rat mammary adenocarcinoma cells using the elastase specific substrates Cbz-Ala-Ala-Pro-Val-6-aminoquinoline and Ac-Ala-Ala-Pro-Ala-7-amino-4-methylcoumarin. This is a sensitive assay which enables rapid (30-120 min) measurement of enzyme activities under conditions of physiological pH and ionic strength and can differentiate between cell-associated and secreted enzyme activities. As the substrates are non-toxic and the method is non-invasive, cells can be reclaimed for further studies. This method thus provides a useful means for screening intact cells for elastase activity. Cell-surface elastase extracts were inhibited by phenylmethylsulphonyl fluoride but not by EDTA, indicating that they are serine proteinases. Extracts also degraded insoluble elastin confirming that these rat mammary adenocarcinoma cells produce elastase.
Grant
A J
AJ
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.
Lerro
K A
KA
Wu
C W
CW
D000230:Adenocarcinoma / Q000201:enzymology; D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003374:Coumarins / Q000378:metabolism; D005470:Fluorometry; D015674:Mammary Neoplasms, Animal / Q000201:enzymology*; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D000634:Aminoquinolines; D003374:Coumarins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase