Studies with digitonin‐treated rat hepatocytes (nude cells)

AbstractIsolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1–2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90–95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment.Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt‐free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15–20 min were required fo