title: Inhibition of cysteine proteinases in lysosomes and whole cells.
abstract: Inhibitors of cysteine proteinases have been used extensively to dissect the roles of these proteinases in cells. Surprisingly though, little work has been performed to demonstrate unequivocally that the inhibitors reach and inactivate their target proteinases in cell culture or in vivo. In the present study, the permeability of lysosomes and whole cells has been studied. Benzyloxycarbonyl (Z)-[125I]iodo-Tyr-Ala-diazomethane (CHN2), an inhibitor of cathepsins L and B, has been shown to label active forms of these enzymes in lysosomes and whole cells. The ability of other cysteine proteinase inhibitors to block this labelling has been used to indicate the permeation of these compounds. All the inhibitors were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomal extracts. In intact lysosomes or cells, however, only N-[N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-leucyl]-3- methylbutylamine ('E-64d') Z-Tyr-Ala-CHN2, Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2. N-[N-(L-3-trans-Carboxyoxirane-2-carbonyl)-L-leucyl]amino-4-gua nidinobutane (E-64) and leupeptin were unable to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomes or in cells. The ability to block labelling in lysosomes is an indication of the ability of the inhibitor to diffuse across membranes. Thus E-64 and leupeptin do not readily permeate membranes and therefore their uptake into cells probably only occurs via pinocytosis.
authors.affiliation: Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: D
authors.initials: D
authors.lastname: Wilcox
authors.forename: R W
authors.initials: RW
authors.lastname: Mason
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002463:Cell Membrane Permeability; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008051:Lipid Bilayers; D008099:Liver / Q000166:cytology; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests
chemical_list: D015853:Cysteine Proteinase Inhibitors; D008051:Lipid Bilayers