Intracellular concentrations, determination

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Acetylcholine is formed from acetyl CoA (produced as a byproduct of the citric acid and glycolytic pathways) and choline (component of membrane lipids) by the enzyme choline acetyltransferase (ChAT). Following release it is degraded in the extracellular space by the enzyme acetylcholinesterase (AChE) to acetate and choline. The formation of acetylcholine is limited by the intracellular concentration of choline, which is determined by the (re)uptake of choline into the nerve ending (Taylor Brown, 1994). 

Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by active transport of choline into the nerve ending 192... 

The induction of this operon responds to the intracellular concentration of cAMP, which is determined by the carbon source available to the cell. When cells are grown on cellobiose or cellulose that do not inhibit adenylate cyclase, cAMP is made in sufficient quantities for induction of cellulase. On the contrary, when cells are grown on glucose or other readily metabolized carbohydrates that do inhibit adenylate cyclase. 

The intracellular concentration of urate in the proximal tubule will ultimately be determined by the balance of influx and efflux. When the transport of urate from the peritubular fluid is high, there is a net elimination of urate across the luminal membrane. In contrast, when the transport of urate from luminal fluid is high, there is a net reabsorption across the basolateral membrane. 

The function of the Cr transporter is assayed using intact fibroblasts, which are cultured for 24 h in a medium that is enriched with either 25 pM or 500 pM Cr [ 18]. The intracellular concentration of Cr in the fibroblasts is determined using the GC-MS procedure described in detail in this chapter (Table 7.2.8). 

Smith, N.R. et al., Differences in the intracellular concentrations of elements in normal and cancerous liver cells determined by x-ray microanalysis, Cancer Res., 38, 1952, 1978. 

Thyroid hormone activity is determined predominantly by intracellular concentrations of (free) T3 in the tissues. This T3 bioavailability depends on 1) the secretion of T4 and T3 by the thyroid, 2) the conversion of T4 to T3 outside the thyroid, 3) the metabolic clearance of T3 and 4) exchange of T4 and T3 between plasma and tissues. It is the purpose of this chapter to review recent advancements in the study of the transport and metabolism of thyroid hormone. Especially the role of deiodination, conjugation and tissue uptake mechanisms will be emphasized in the regulation of thyroid hormone action. 

For in vitro assays, it is extremely difficult to determine the relevant intracellular concentrations of substrates due to compartmentalization of particular reactions, competing reactions that produce and consume common substrates, and metabolic channeling (Albe et al, 1990 Srere, 1987). Consequently, in vitro kinetic measurements may not be very useful for assessing in situ or in vivo reaction rates. However, provided assays conditions are carefully controlled, kinetic parameters may be very useful for characterizing enzyme isoforms and enzymes from different metabolic pools, different organisms, or from organisms collected from different environments. 

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