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Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells
Abstract:
The distribution of membrane-bound receptors and enzymes between the cell surface and the cell interior can be determined without solubilization or gross disruption of cell organelles in the presence of the nonionic detergent digitonin. This steroid glycoside permeabilizes cells, releases cytoplasmic proteins with subunit molecular weights up to 200,000, and allows exogenous molecules to gain access to intracellular receptors. All cell types examined were affected similarly by digitonin. Permeabilization was complete within 2 min at 0 degree C and did not require the continued presence of digitonin. A characteristic amount of protein (approximately 50%) was lost between 0.02 and 0.08% (w/v) digitonin. Three independent systems were examined: the insulin receptor in 3T3 fibroblasts and the asialoglycoprotein receptor and the Na+/K+-ATPase in rat hepatocytes. In each case an increase in the specific activity of enzyme/receptor occurred over a range of detergent concentration in which the retention of cell protein was constant and virtually no solubilization of membrane-bound activity occurred. The binding of 125I-asialo-orosomucoid to rat hepatocytes at 0 degree C in the presence of digitonin was linear with cell number and kinetically indistinguishable from binding to intact cells. Receptors exposed by digitonin were shown to be intracellular by light microscopic examination of permeabilized cells first treated with antiserum to the receptor and then with a second antibody horseradish peroxidase conjugate. The use of digitonin has many advantages over procedures which require total cell disruption or solubilization to assess intracellular receptors. The technique has already been valuable in studies on recycling and endocytosis mediated by the asialoglycoprotein receptor (P.H. Weigel and J.A. Oka (1983) J. Biol. Chem. 258, 5095-5102) and should also be useful in studies with other membrane-bound receptors and enzymes in other cell types.