Digitonin 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...

Degradation of plasmid DNA is activated by divalent cations such as Ca2+, Mn2+ and Mg2+ in the cytosolic extract obtained by digitonin permeabilization and monitored by Southern blot analysis and 32P-... 

Wilson, G.L., Dean, B.S., Wang, G. and Dean, D.A. (1999) Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J. Biol Chem., 274, 22025-22032. 

Fiscum, G. 1985. Intracellular levels and distribution of Ca2+ in digitonin-permeabilized cells. Cell Calcium 6 25. 

Terbush DR, Holz RW (1986) Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells. J Biol Chem 261 17099-106... 

In chicken granulosa cells, TMB-8, a putative antagonist of intracellular calcium mobilization, inhibited progesterone synthesis in calcium free medium and also inhibited LH stimulated 45Ca release from digitonin permeabilized cells [31]. The latter occurred in two phases after 1-2 min followed by a slower phase after 5 min. Forskolin and 8-Br-cyclic AMP stimulated only the slow phase of calcium mobilization. These studies indicate that LH may initially stimulate calcium mobilization in granulosa cells via a cyclic AMP-independent pathway followed by a slower cyclic AMP-dependent pathway. 

Bittner MA, Holz RW (1988) Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells. In J. Neurochem. 51 451-6... 

Bittner MA, DasGupta BR, Holz RW (1989 a) Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells. In J. Biol. Chem. 264 10354-60... 

Bittner MA, Habig WH, Holz RW (1989 b) Isolated light chain of tetanus toxin inhibits exocytosis studies in digitonin-permeabilized cells. In J. Neurochem. 53 966-8... 

Early work on the mechanism of nuclear import focused on proteins containing a basic NLS, similar to the one in SV40 large T-antlgen. A digitonin-permeabilized cell system provided an in vitro assay for analyzing soluble cytosolic components required for nuclear Import (Figure... 

Adeli, K., Wettesten, M., Asp, L., Mohammadi, A., Macri, J., Olofsson, S.-O. 1997. Intracellular assembly and degradation of apolipoprotein B-lOO-containing lipoproteins in digitonin-permeabilized HepG2 cells. 

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. 

Fig. 20. Oligosaccharide-mediated nuclear transport of glyco-QDs. (A) Chemical structures of neoglycoconjugates. (B) Synthesis of glyco-QDs by thiol exchange reaction. (C) Confocal fluorescence images (top panels) and differential interference contact (lower panels) of digitonin-permeabilized HeLa cells incubated with glyco-QDs. (D) Digitalized fluorescence intensity of different QDs in the nucleus. Adapted from Ref 204. (See Color Plate 42.)...

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. 

In the present work we have studied effects of 3-thia fatty acids on P-oxidation and CPT I activity in freshly cultured (4-6 hours) rat hepatocytes. By using digitonin-permeabilized cells we were able to measure CPT I activity directly in hepatocytes which are possibly a more physiologically relevant system than isolated mitochondria. 

Incubate coverslips on 50 pL-drops of Alexa Huor 488-conjugated anti-mouse secondary and Cyanin 5-conjugated anti-rabbit secondary antibody dilutions (1 500) for 40 min at room temperature. This step allows the detection of cytoplasmic bacteria and calnexin at the cytoplasmic face of the ER that have been recognized following digitonin permeabilization. 

Adam, S. A., Sterne-Marr, R., and Gerace, L. (1991). In vitro nuclear protein import using digitonin permeabilized mammalian cells. Methods Cell Biol. 35, 469-481. 

Diaz, R., and Stahl, P. D. (1989). Digitonin permeabilization procedures for the study of endosome acidification and function. Methods Cell Biol. 31, 25-43. 

Dihydroorotate dehydrogenase (EC 1.3.99.11) catalyses the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. Inhibition of cytochrome c oxidase indirectly inhibits dihydroorotate dehydrogenase activity. In digitonin-permeabilized cells, sodium l,l-diethyl-2-hydroxy-2-nitroso-hydrazine, a chemical nitric oxide donor, induced a dramatic decrease in dihydroorotate-dependent O2 consumption (Beuneu et al. 2000). The inhibition was reversible and more pronounced at low O2 concentration it was correlated with a decrease in orotate synthesis. 

Table 4. Label in phosphate esters released from digitonin-permeabilized soybean cells pre-labelled from [ H]-inositol...

Chang D, Dawson DC (1988) Digitonin-permeabilized colonic cell layers. J Gen Physiol 92 281-306... 

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