Differential lysis of plasma membranes and granules of human neutrophils by digitonin.
The effect of the detergent digitonin on lysis of granule and plasma membranes of human neutrophils was studied. Either linear or sigmoid dose-response for release of the cytoplasmic marker lactate dehydrogenase and the granule markers lysozyme, beta-glucuronidase, lactoferrin, and myeloperoxidase was noted using digitonin concentrations ranging from 0.001 to 0.1 mM. However, release of the cytosol compartment was far more sensitive to the detergent than the granule compartment, with more release of lactate dehydrogenase than of lysozyme at 0.01-0.08 mM digitonin. Distinction between the two compartments was optimal at 0.025 mM digitonin. By examining in parallel the digitonin-induced release of exogenous fluorescent or luminescent indicators, a granule location was demonstrated for the pH indicator 9-aminoacridine, while the calcium probes aequorin and Quin 2 were released coincident with release of the cytosolic enzyme lactate dehydrogenase. These findings were employed to validate use of the indicators for monitoring of ion translocation in the intact cell. The differential effect of this detergent on subcellular membranes provides a broadly applicable technique for rapid assessment of the subcellular localization of tracer substances. Rapid monitoring may help to avoid problems of redistribution during cell fractionation.
D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009113:Muramidase / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure
D007770:L-Lactate Dehydrogenase; D009113:Muramidase; D004072:Digitonin