The interactions of protein inhibitors with tumour proteases studied in solution and immobilised on cell surfaces in frozen sections.
The cell surface protease guanidinobenzoatase (GB) has been purified from human colonic and lung carcinoma tissue by an affinity step involving the binding of the enzyme either onto fibrin fibrils or onto agmatine-sepharose. The inhibitor protein (I) was extracted from the cytoplasm of tumour cells and isolated by an affinity step involving the binding of I to GB on the surface of cultured carcinoma cells. The interaction of GB and I in solution was followed by kinetic studies employing the release of the fluorescent 4-methylumbelliferone (MU) from the synthetic substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUGB). The interaction of soluble I with membrane bound GB was followed by using the yellow fluorescent probe 9-aminoacridine (9AA) which binds to active GB but not to GB-I. The results of these studies demonstrated the presence of isoenzymic froms of GB which were recognized specifically by their appropriate isoinhibitor, isolated from the appropriate cell type. This high degree of selectivity suggests a cell specific regulatory role for the inhibitors and the possibility that they might be used for the delivery of cytotoxic molecules to the surface of specific types of tumour cells.
Steven
F S
FS
School of Biological Sciences, University of Manchester, U.K.
Anees
M
M
Talbot
I C
IC
Blakey
D C
DC
Hasleton
P S
PS
D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000473:pathology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003106:Colon / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000201:enzymology* / Q000473:pathology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004800:Enzymes, Immobilized; D011480:Protease Inhibitors; D002265:Carboxylic Ester Hydrolases; C037288:guanidinobenzoate esterase; D010450:Endopeptidases