Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis.
Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.
D000818:Animals; D002279:Carcinoma 256, Walker / Q000201:enzymology* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002471:Cell Transformation, Neoplastic / Q000032:analysis*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D008297:Male; D009283:Naphthol AS D Esterase / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases
D011092:Polyethylene Glycols; D017830:Octoxynol; D004950:Esterases; D009283:Naphthol AS D Esterase; D010450:Endopeptidases; D012697:Serine Endopeptidases