Intracellular milieu

Godt, R.E. Nosek, T.M. (1989). Changes of intracellular milieu with fatigue of hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. J. Physiol. 412, 155-180. 

Why mammalian ferritin cores contain ferrihydrite-like structures rather than some other mineral phase is less easy to understand, and presumably reflects the way in which the biomineral is built up within the interior of the protein shell together with the geometry of the presumed nucleation sites. The phosphate content in the intracellular milieu can readily be invoked to explain the amorphous nature of the iron core of bacterioferritins and plants. Indeed, when the iron cores of bacterioferritins are reconstituted in the absence of phosphate, they are found to be more highly ordered than their native counterparts, and give electron diffraction lines typical of the ferrihydrite structure. Recently it has been reported that the 12 subunit ferritin-like Dps protein (Figure 19.6), discussed in Chapter 8, forms a ferrihydrite-like mineral core, which would seem to imply that deposition of ferric oxyhydroxides within a hollow protein cavity (albeit smaller) leads to the production of this particular mineral form (Su et al., 2005 Kauko et al., 2006). 

All aerobic organisms contain substances that help prevent injury mediated by free radicals, and these include antioxidants such as a-tocopherol and the enzymes superoxide dismutase and glutathione peroxidase. When the protective effect of the antioxidants is overwhelmed by the production of reactive oxygen species, the intracellular milieu becomes oxidative, leading to a state known as oxidative stress (Halliwell and Gutteridge, 1999). Thus the balance between the generated free radicals and the efficiency of the protective antioxidant system determines the extent of cellular damage. 

Antioxidant composition of the intracellular milieu is different than that of extracellular fluids. Therefore, measurement of TAC of tissue homogenates may yield valuable information, complementary to that obtained from studies of blood plasma and other body fluids (see Table 11). 

One physical consequence of the excluded volume effect is that solutions crowded with macromolecules tend to be highly nonideal. In an ideal solution, a particle has access to any region of the solution. This is clearly not true for the intracellular milieu (figure 6.22). As a result of excluded volume effects, the chemical activities of both large and small solutes are considerably higher than their actual concentrations (this is another aspect of the nonideality of the intracellular milieu). The nonideal characteristics of the intracellular solution favor a minimization of excluded volume, that is, they... 

The phenomenon of molecular crowding and the consequent excluded volume effects that arise from crowding raise important issues concerning protein structure and function in vivo. Other things being equal, one would predict that proteins would be more stable within their intracellular milieu than in dilute solutions—strictly because of excluded volume effects. If increased stability leads to reduced rate of function, then in vitro studies of enzymatic activity (kcat values in particular) may be providing a misleading picture of what occurs in the cell. 

The joint evolution of proteins and the solution in which they occur is an issue within evolution that we treat at numerous junctures in this volume. The types of effects noted for enzymes of M. fervidus and paralogs of MDH show that joint evolution of proteins and solutes may be of broad importance in adaptation to temperature. Data such as these also have a practical lesson for us they show that the complexity of the intracellular milieu must be carefully evaluated when designing and interpreting experiments done in vitro. All partners in the evolutionary process must be considered if the nature of physiological adaptation is to be appreciated. This point brings us to one of the most striking differences exhibited by proteins under in-vitro versus in-vivo conditions the extent to which the primary structure of the protein is able to direct its folding into the... 

As discussed earlier in this chapter and also in chapter 6, thermal stabilities of proteins in vivo are influenced by many constituents of the intracellular milieu, including low-molecular-mass protein stabilizers. The process of protein folding, whether during initial synthesis or following heat-induced unfolding, thus will be influenced not only by activities of protein chaperones, but also by the activities of low-mole-cular-mass organic solutes. In principle, heat stress could be ameliorated in part by accumulation of low-molecular-mass protein-stabilizing solutes that favor formation of the compact, folded state of proteins. Such chemical chaperones could complement the activities of protein chaperones. 

Another major factor when considering whole-cell versus cell-free reactions are the overall reaction kinetics. Some enzymatic reactions utilize a complex multicomponent enzyme system. Reconstitution of the crude or purified enzyme components are not usually as effective in vitro as they are when they remain in the intracellular milieu. Whole cells have often been called little bags of enzymes. Although this is an oversimplification, it is a useful concept to consider. Whole cells sequester the enzyme components in a small but concentrated form, which is usually optimal for high efficiency. Whole cells also contain co-factors, including the systems that recycle them, and control pH and ionic strength. Altogether these factors combine to make whole cells a very useful form for the presentation and use of sensitive enzyme catalysts. 

High concentrations of heavy metals that affect, directly or indirectly, the redox potential in the reducing intracellular milieu, particularly copper, induce the formation of reactive oxygen species. These, in turn, can trigger the chain reaction causing lipid peroxidation, which affects membrane integrity and induces structural changes to proteins and nucleic acids that can result in cell death. 

Upon entry into bacterial cells, the disulfide bond in compound 3 is reduced by sulfydryl compounds present in the intracellular milieu, resulting in the formation of thiol... 

Until now, we have dealt solely with changes to the enzyme molecule as the mechanism of adaptation, but enzyme function and stability can also be strongly influenced by the composition of the intracellular milieu in which the enzyme... 

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