2.4.5.1—
Evidence from Kinetics

One widely used approach to gather information about carriers has been the application of kinetics first derived from enzyme reactions. The evidence for these carriers is obtained by placing a cell or tissue in a range of solute concentrations and measuring the initial rate of uptake. As illustrated in Fig. 2. 15 the uptake rate shows a tendency to saturate at higher concentrations and can thus be used to calculate the maximum velocity, V_max , possible under the conditions used in the experiment. By making a double reciprocal plot of the data (Fig. 2.15) the concentration of solute at which half maximal velocity is achieved can

Figure 2.15
Saturation kinetics of solute uptake versus concentration. Such results are used
as evidence for the association of the solute and a carrier. The double reciprocal plot of
the data gives a more accurate estimate of  V_max  and K_m  when the number of points is limited.

be estimated. This is known as the Michaelis Constant, K_m . The K_m measures the affinity of the carrier for the solute it carries; if the affinity is high then the concentration, K_m will be low and vice versa. For most ions in plant tissue K_m is quite low, concentrations ranging from 5–100 mM , but for sugars and other metabolites K_m values are usually greater than 300 mM . Much work of this kind is summarized by Epstein (1972) who shows that at concentrations less than 0.1 mM , the uptake of a given ion is not subject to serious interference from other

common ions in solution. There is, however, competitive inhibition between related ions of similar molecular dimensions, e.g. K⁺ uptake is inhibited competitively by Rb⁺ but not by Na⁺ ; Ca²⁺ is inhibited by Sr²⁺ but not by Mg²⁺ . Thus the carriers which bind the major nutrient ions at low concentrations appear to be highly ion-selective. At higher concentrations (more than 1.0–10.0 mM ) this selectivity begins to decline. The interpretation of this observation is contentious and beyond the scope of this chapter but can be pursued in Epstein (1972), Laties (1969) and Clarkson (1974).

The limitation of the kinetic approach is that it can tell us nothing about the nature of the carrier. One can observe similar uptake kinetics for ions whose transport into the cell must be mediated by ion pumps e.g. H₂ PO₄ ^– and Cl^– (see p. 48) as for ions which probably diffuse into the cell passively e.g. Na⁺ and Ca²⁺ and for those which are completely exotic and toxic, e.g. Tl⁴⁺ (Barber, 1974). Indeed, it has been pointed out that saturation kinetics of this kind would also be found if salt movement was observed across a synthetic membrane containing nothing but pores (Stein & Danielli, 1956), where the system would saturate when all of the pores were filled with solute at any moment in time; V_max is, after all, merely a measurement of capacity to react or transport and K_m is derived from it (Fig. 2.15).