Characterization of isolated rat-liver cells made permeable with filipin

When isolated rat-liver cells were incubated for 1 min at 37 degrees C with filipin at a concentration of 50 microM, the plasma membrane became permeable to sucrose, inulin, glycerol 3-phosphate and other low-molecular-weight compounds. Upon removal of the filipin and subsequent incubation of the cells at 37 degrees C there was a gradual leakage of lactate dehydrogenase from the cells. However, the leakage of lactate dehydrogenase could be prevented for about 10 min by including glutathione and ATP in the incubation medium. The filipin-treated cells were able to metabolize phosphorylated sugars. The conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, glucose-6-phosphate and glucose was inhibited by AMP but not by high concentrations of fructose 1,6-bisphosphate. The results indicate that filipin-treated cells can be used to study the kinetic parameters of enzymes in their macromolecular environment in situ.