Kinetics of L-valine uptake in tobacco leaf discs. Comparison of wild-type, the digenic mutant Valr-2, and its monogenic derivatives

Abstract

Uptake rates of L-valine in epidermis-free leaf discs of tobacco (Nicotiana tabacum L. cv. Xanthi) were measured over the concentration range 0.1 μM to 50 mM. Wild-type tobacco was compared with the digenic mutant Val(r)-2 (genotype vr2/vr2; vr3/vr3), and with the monogenic mutant strains h9 and h10 (genotype +/+; vr3/vr3) and h17 and h23 (genotype vr2/vr2; +/+). Rate equations consisting of one to three Michaelis-Menten terms, possibly in combination with a linear term were fitted to the kinetic data. These rate equations are equivalent to rational polynomials which may be regarded as the general type of mathematical function describing the kinetics of enzymes and carriers. Kinetic data of the four genotypes conformed to the sum of three Michaelis-Menten terms. Accordingly, three kinetic components could be distinguished. In the wild-type the approximate Kms were 40 μM, 1mM, and 40 mM, respectively. In Val(r)-2 a component with a very low Km (about 4 μM) was found which may represent either the modified low-Km component of the wild-type or a fourth component which is undetectable in the wild-type by kinetic analysis. The Vmax of the low-Km component in Val(r)-2 was at least a 100-fold lower than in the wild-type. In the presence of one of the mutant genes the calculated Vmax of the low-Km component was 48% (strains h9 and h10) or 40% (strains h17 and h23) of the corresponding Vmax in the wild-type. It is reasoned that the mutations have no effect on the activity of the other two kinetic components, though the evidence for this is circumstantial. Autoradiographs of leaf discs showed that in Val(r)-2 the uptake of (14)C-labelled valine in both mesophyll and minor veins was strongly reduced as compared with the wild-type.