Cell membrane Ionic milieu

Pores formed by PFTs are usually fairly well defined in size, so that judicious choice of toxin and of experimental conditions will generally enable one to produce stable membrane lesions ranging from approximately 8 A to 300 A in functional diameter. It becomes possible to manipulate the intracellular ionic milieu, to introduce small molecules such as nucleotides into the cells, or to apply large molecules such as antibodies to the cytoplasm. Since pure preparations of PFT are devoid of enzymatic (proteolytic) activity, they cause no direct alterations of cell constituents. In the following, simple strategies for utilization of PFTs will be outlined. 

In many cases, enzymes of interest are associated with insoluble parts of the cell, such as mitochondria, endoplasmic reticulum or membranes, and an extraction procedure is necessary. Not all of these enzymes can survive solubilization in the absence of their normal cellular milieu. The extraction buffers should not only have the correct pH and ionic strength but also the appropriate stabilizers should be added (Table 5.17). 

Non-ionic diffusion can also produce unwanted effects, as in the case of aspirin (acetylsalicylic acid figure 2.8b). In the acidic milieu of the stomach, this molecule will be protonated and thus uncharged, which promotes its diffusion into the cells of the stomach mucous membrane. Inside the cell, the pH is very close to neutral, which will lead to deprotonation of aspirin. Diffusion of the deprotonated (charged) form out of the cell will be much slower than entry, so that aspirin will accumulate inside the cells to con-... 

In all cells, the plasma membrane acts as a permeability barrier that prevents the entry of unwanted materials from the extracellular milieu and the exit of needed metabolites. Specific membrane transport proteins In the plasma mem brane permit the passage of nutrients Into the cell and metabolic wastes out of It others function to maintain the proper Ionic composition and pH ( 7.2) of the c3d osol. The structure and function of proteins that make the plasma membrane selectively permeable to different molecules are discussed In Chapter 7. 

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