Digitonin membrane permeabilization

Fig.3.8.3 Oxygen uptake by intact (a) and digitonin-permeabilized (b) fibroblasts. I-V Respiratory chain complexes I-V, AcCoA acetylcoenzyme A, BSA bovine serum albumin, CCP carbonyl cyanide m-chlorophenylhydrazone, Cit citrate, CoA coenzyme A, CS citrate synthase, Dig digitonin, Fo FI the ATPase components, Fum fumarase, G3P glycerol-3-phosphate, im inner membrane, Mai malate, Malo malonate, MDH malate dehydrogenase, OAA oxaloacetate, om outer membrane, PDH pyruvate dehydrogenase, Pi inorganic phosphate, Pyr pyruvate, Q ubiquinone, Rot rotenone, Succ succinate, t time...

Permeabilizes membranes e.g. mitochondrial digitonin particles cholesterol interaction [antifungal, detergent, haemolytic]... 

Digitonin a mild detergent that permeabilizes the plasma membrane of most mammalian cells without affecting organelles such as the mitochondria... 

Organic solvents like methanol or acetone also can be used to open membranes, but they also act as denaturing fixatives. Solvents are used at -20 C for 10 min, but the use is not recommended. As described in previous sections, denaturing fixatives may cause loss of epitopes in the cells. Other detergent-like agents, such as saponin or digitonin, are used for transient permeabilization of cell cultures, and reversibly insert into and out of plasma membranes next to cholesterol. These agents must be present in all solutions after the fixative and should only be used for electron microscopy immunocytochemistry. 

Vercesi, A. E., Bernardes, C. F., Hoffman, M. E., Gadelha, F. R. and Docampo, R. (1991) Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ. J. Biol. Chem. 266 14431-14434. 

Guzman, M. Geelen, M.J.H. (1992) Biochem. J. 287, 487-492. Activity of camitne palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid. 

For immunolabeling purposes as well as nucleocytoplasmic transport studies, monolayers of cultured cells can be easily permeabilized by treatment with digitonin (Adam et ai, 1990). The rationale behind the method is that digitonin more or less selectively attacks membranes containing cholesterol (such as the plasma membrane) but not membranes containing no or very little cholesterol (such as the nuclear membranes). We use the following method to permeabilize 3T3 cells ... 

Cells other than 3T3 cells can be permeabilized by the same method. However, the optimal digitonin concentrations and incubation times should be determined for each cell line separately. Of course, cells can be also permeabilized by many detergents other than digitonin, such as Triton X-100 and saponin. However, these reagents not only permeabilize the plasma membrane but also the nuclear envelope. It is also uncertain whether the NPC is damaged and nucleoporins are removed by these detergents. 

Treatment with 25 //g/ml digitonin for 5 min does not permeabilize the peroxisomal membrane (differential permeabilization). Only epitopes on the cytosolic surface of the peroxisomal membrane can be detected. 

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