, 2012 and Leroux et al., 2013). During the development of the in vivo-like assay media for the various organisms, similar challenges were faced in each study. The most common challenges will be addressed here. These are (i) the buffer capacity and anion composition of the medium; (ii) macromolecular crowding; and (iii) the effect of pH. In all studies on the development of an in-vivo-like assay medium the buffer capacity was one of the most important issues coming forward. A buffer is

needed, since the added components as well as the altering reactant concentrations may affect the pH in the assay. The buffer capacity of cells can be ascribed mainly to inorganic phosphate, amino acids and amino-acid side chains in

proteins ( Castle et al., 1986) (but see Poznanski et al., 2013). However, inorganic p38 kinase assay phosphate is also an effector of many enzymes, as for instance the glycolytic enzyme pyruvate kinase in L. lactis ( Goel et al., 2012). Therefore, inorganic phosphate can only be used when it is in reality high in the cells. AZD6244 nmr Indeed, Wu et al. (2006) reported that S. cerevisiae had a high intracellular concentration of high phosphate due to the high phosphate in the medium. In the case of L. lactis, however, intracellular phosphate was low and therefore Goel et al. (2012) decided to use the non-physiological HEPES buffer instead. The use of a non-physiological buffer, such as HEPES or PIPES, is not preferable, since it adds a compound to the medium that is not present in the cell. Yet, in cases like described above it seems the best alternative, as long as the non-physiological compound does not affect the enzyme kinetics. In this respect, van Eunen et al. (2010) showed that the use of PIPES instead of glutamate does not affect the activity of the yeast glycolytic enzymes. Even when phosphate can be used as a buffer at its physiological concentration, it is important to keep in mind that intracellular phosphate may fluctuate upon environmental changes. Another issue in developing an in vivo-like assay medium is whether and how to mimic the effect of macromolecular crowding. Macromolecular crowding can alter

the properties of enzymes in vivo ( Ellis, 2001, Garner and Burg, 1994 and Zimmerman and Minton, 1993). For instance, learn more the cytosol of E. coli contains around 300–400 mg/ml macromolecules ( Zimmerman and Trach, 1991). If this intracellular crowding effect is not taken into account, enzymes may behave in a different way in in vitro assays ( Minton, 2006). For instance, Rohwer et al. (1998) showed that the flux through the phosphotransferase system (PTS) in E. coli depends on the presence and concentration of macromolecules. They used up to 9% of polyethylene glycol (PEG), an inert macromolecule, to mimic the intracellular crowded environment. This altered the strength of protein–protein interactions, which is an important factor in the kinetics of the PTS.