Temporary competitive inhibition of a tumour cell surface protease as a protective mechanism in the preparation of the membrane bound native enzyme in the presence of excess cytoplasmic inhibitors.
Tumour cells possess a cell surface protease which is recognised and inhibited by a cytoplasmic protein extractable from frozen sections of tumour cells. In order to prepare sections with tumour cells carrying cell surface-bound native protease in the absence of this internal inhibitor we have used a reversible competitive inhibition step as a temporary measure to protect the active centre of GB whilst the cytoplasmic inhibitor is extracted from the frozen sections. These sections are described as protected in the sense that the enzyme is native and fully functional now that potential inhibitors have been extracted. The protected cell surface protease immobilised in the cell surface of squamous cell carcinoma cells has been used as the target for inhibition studies and displacement studies. The ability to follow these inhibition and exchange reactions concerning the cell surface protease has been made possible by virtue of the fluorescent probe, 9-amino acridine, which locates the active centre of the protease. Cells with active protease bind 9-amino acridine and fluoresce yellow; cells lacking this protease or having inhibited protease fail to bind 9-amino acridine and do not fluoresce.
Steven
F S
FS
Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, UK.
Griffin
M M
MM
Maier
H
H
D000585:Aminacrine; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000473:pathology; D002462:Cell Membrane / Q000201:enzymology*; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D009363:Neoplasm Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D000585:Aminacrine; D010450:Endopeptidases