title: Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells.
abstract: Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01-0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.
authors.affiliation: Biochemical Engineering Institute, Saarland University, D-66123 Saarbrücken, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Niklas
authors.forename: Armin
authors.initials: A
authors.lastname: Melnyk
authors.forename: Yongbo
authors.initials: Y
authors.lastname: Yuan
authors.forename: Elmar
authors.initials: E
authors.lastname: Heinzle
mesh_terms: D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D004072:Digitonin / Q000737:chemistry; D004798:Enzymes / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry
chemical_list: D004798:Enzymes; D017830:Octoxynol; D005954:Glucosephosphate Dehydrogenase; D004072:Digitonin