Cell permeabilization, protein release

Bader MF, Thierse D, Aunis D etal. (1986) Characterization of hormone and protein release from alphatoxin-permeabilized chromaffin cells in primary culture. J. 

An important factor complicating the recovery of recombinant proteins from Escherichia coli is their intracellular location. An alternative to the commonly used method of releasing these proteins by mechanical disruption is to chemically permeabilize the cells. The objective of this research was to characterize the protein release kinetics and mechanism of a permeabiliza-tion process using guanidine-HCl and Triton-XIOO. The protein release kinetics were determined as a function of the guanidine, Triton, and cell concentrations. Some of the advantages over mechanical disruption include avoidance of extensive fragmentation of the cells and retention of the nucleic acids inside the cell structure. 

Intrinsic (mitochondrial) pathway of caspase activation is initiated by the permeabilization of the mitochondrial outer membrane by proapoptotic members of the Bcl-2 family, resulting in a release of cytochrome c and other proteins from the intermembrane space of mitochondria into the cytosol. Cytochrome c translocation to the cytosol may follow a number of possible mechanisms. However, once in the cytosol, cytochrome c binds to apoptosis protease activating factor (Apaf-1) and in the presence of dATP or ATP facilitates Apaf-1 oligomerization and the recruitment of procaspase-9. The formation of this caspase-activating complex, termed the apoptosome, results in the activation of procaspase-9, and this in turn cleaves and activates the effector caspase-3 and -7. Activated effector caspases cleave key substrates in the cell and produce the cellular and biochemical events characteristic for apoptosis [33-35]. 

The response of mast cells includes release of arachidonic acid due to membrane PhL A2 activation following a ligand-induced increase in cellular Ca2+. PTX reduces the Ca2+-mediated GTP S-dependent release of this fatty acid in permeabil-ized cells [102,103]. This raised the possibility of a direct link, not only between receptors and PhL C, but also between receptors and PhL A2. Existence of a G protein-mediated. PTX-sensitive, activation of PhL A2 independent of G protein-mediated activation of PhL C was confirmed in studies first with fibroblasts [104] and then with FRTL thyroid cells [105]. Studies with the latter cells show that a,-adrenergic receptors promote arachidonic acid release [105] and that this effect is mimicked in permeabilized cells by GTP and is not blocked by inhibition of PhL C with neomycin. Thus, at least two Gp proteins need to be defined a Gp-stimu-lating PhL C (Gp(c) and a Gp-stimulating PhL A2 (Gp,a). It is possible that rat brain G is PTX-sensitive Gplc. 

Data in Table IV show the permeabilizing effect of chitosan expressed in release of total proteins when chitosan was used as the immobilizing agent in complex coacervate capsules, but suggest low cell viability as measured by respiration. However, Beaumont and Knorr (10) reported that fresh weight of cells recovered at the end of a culture period in the growth medium is a better indicator of actual cell viability than respiration data. 

Cell envelope (cell wall, outer membrane) Glutaraldehyde EDTA, other permeabilizers Cross-links proteins Gram-negative bacteria removal of Mg2+, release of some LPS... 

Ahnert-Hilger G, Brautigam M, Gratzl M (1987) Ca -stimulated catecholamine release from permeabilized PC 12 cells Biochemical evidence for exocytosis and its modulation by protein kinase C and G-proteins. Biochemistry 26 7842-7848. 

For each BoNT serotype, the dichain form constimtes the active configuration of the neurotoxin the isolated LC and HC are devoid of systemic toxicity. The absence of toxicity is consistent with findings that the LC cannot gain access to the cytosol unless it is coupled to the HC and that the HC lacks the ability to inhibit neurotransmitter release (Stecher et al., 1989 Goodnough et al., 2002). The isolated LC does, however, remain enzymatically active as evidenced by its ability to inhibit exocytosis from permeabilized chromaffin cells (Stecher et al., 1989), by its ability to cleave SNARE proteins in cell-free assays (Adler et al., 1998), and by its capacity to inhibit ACh release in skeletal muscle when delivered by liposomes (de Paiva and Dolly, 1990). It is not clear whether any portion of the HC is translocated along with the LC, and if so, whether it exerts a role in enhancing the catalytic activity or stability of the LC. 

From these results, three major differences between chemical permeabilization and mechanical disruption can be identified. First, the release occurs by fundamentally different mechanisms. With mechanical disruption the cells are essentially torn apart, whereas with chemical treatment the cell structure is still present but has been altered to allow release of intracellular components. Second, there is a nearly complete preferential release of protein over DNA. Third, there is a partial selective release of protein over RNA. This selectivity may result from a molecular sieving mechanism. The average protein molecular weight is 40,000 whereas the cellular DNA has a molecular weight of 2.5 x 10s (12). The molecular weight distribution of RNA 18% is 25,000, 27% is 500,000, and 55% is 1,000,000 is such that most of the RNA is also significantly larger than proteins (12). 

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