Pore Permeabilization

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

D.D. Kindler, C. Thiffault, N.J. Solenski, J. Dennis, V. Kostecki, R. Jenkins, P.M. Keeney and J.P.Rr. Bennett, Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore, Mol. Cell. Neurosci., 23 (2003) 559-573. N.R. Ferreira, A. Ledo, J.G. Frade, G.A. Gerhardt, J. Laranjinha and R.M. Barbosa, Electrochemical measurement of endogenously produced nitric oxide in brain slices using Nafion/o-phenylenediamine modified carbon fiber microelectrodes, Anal. Chim. Acta, 535 (2005) 1-7. 

Membrane destabilization can be simultaneously monitored by using propidium iodide. This non fluorescent and poorly permeant small molecule enters into the cells only upon membrane permeabilization/ destabilization and becomes fluorescent upon binding to DNA in the nucleus. In the presence of melittin, cells are also rapidly (200 s) red-labeled upon peptide induced membrane permeabilization (Figure 16.4D). It can be seen that the diffusion of propidium iodide inside the cells is correlated with the leak of EGFP. The results show that melittin induces the formation of pores in the plasma membranes allowing the passage of... 

Application of an electric field to lipid bilayers such as those found in cellular membranes causes short-term depolarization of the membrane and formation of pores and other structural changes [17]. These so-called electropores allow the uptake of hydrophilic macromolecules such as plasmid DNA, siRNA, or proteins that are otherwise unable to diffuse passively through this highly regulated barrier. The use of high-voltage electrical pulses to permeabilize cell membranes was first described as a tool to deliver DNA into mammalian cells in 1982 (Wong and Neumann 1982 Neumann et al. 1982). In cuvette-based methods, cells are... 

Based on the results of a-LTX mutagenesis, strong correlation exists between pore formation and stimulation of Ca2+-dependent exocytosis from neuroendocrine cells. However, in some experiments with chromaffin cells, a-LTX action does not involve Ca2+ entry (Michelena et al. 1997). In addition, a-LTX sensitizes chromaffin cells to Ca2+ even when the cells are permeabilized and toxin pores should have no effect this involves protein kinase C (PKC) activation (Bittner and Holz 2000). Furthermore, the ability of a-LTXN4C to induce Ca2+-dependent exocytosis without forming pores implicates a stimulating mechanism other than pore formation. 

Figure 17.7. Two types of mitochondrial membrane permeabilization. Upper Bax/Bak pore leads to release of intermembrane space proteins, but the inner membrane is intact. Lower PTP (permeability transition pore) opening destroys the impermeability of the inner mitochondrial membrane (IMM). The pore opening causes influx of solutes and water into the matrix resulting in swelling. The mitochondrial swelling ruptures outer mitochondrial membrane (OMM). CypD, cyclophilin D.

Permeabilize membranes (bind cholesterol, form pores) [antibiotic, haemolytic]... 

The dose-dependence of pore-forming activity for SLO is rather steep. Therefore prepare a series of 1 1 dilutions of SLO in LC. As outlined below, wash the cells with KRH, add 50 xl of your SLO dilutions to the wells for 7 min, aspirate and add trypan blue working solution. Leave the solution for 10 min (for reproducibility, the time of incubation with SLO and with trypan blue is important). Check the degree of permeabilization under the light microscope. The correct dilution of SLO is characterized by 100% of blue cells and minimal loss of cells. 

Structural homologies between PFTs and other toxins have not been identified. However, the process of membrane permeabilization may be operative in many cases where proteins have to escape from an intracellular compartment. Well known examples are diphtheria toxin, the neurotoxins and anthrax toxin. Specific domains in many intracel-lularly active toxins have in fact been shown to produce pores in artificial lipid bilayers, and membrane permeabilization is thought to form the basis for translocation of the active moieties from the late endo-some to the cytoplasm (reviewed in Montecucco et ai, 1994). The molecular mechanism of this translocation remains obscure. In the... 

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