title: Direct evidence for the cell surface location of a protease-inhibitor complex on intact leukaemia cells.
abstract: The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, U.K.
authors.forename: F S
authors.initials: FS
authors.lastname: Steven
authors.forename: M M
authors.initials: MM
authors.lastname: Griffin
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000648:ultrastructure*; D010450:Endopeptidases; D005456:Fluorescent Dyes; D007942:Leukemia, Experimental / Q000378:metabolism* / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods; D011480:Protease Inhibitors / Q000032:analysis*; D051381:Rats
chemical_list: D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D002265:Carboxylic Ester Hydrolases; C037288:guanidinobenzoate esterase; D010450:Endopeptidases