Kinetic analysis of 75selenium uptake by mitochondria of germinating Vigna radiata of different selenium status.

Abstract

Earlier studies in our laboratory demonstrated the beneficial role of Se in Vigna radiata, a Se-deficient legume, during germination, as reflected in growth-related parameters and specific uptake of 75Se. Uptake of Na2(75)SeO3, added in vitro by mitochondria isolated from seedlings germinated in control (without Se), and Se-supplemented groups (0.5, 1.0, and 2.0 ppm Se) indicated a proportional increase in the uptake with added Na2(75)SeO3, in concentrations up to 25 microM. The uptake of 75Se, increased linearly with time up to 15 min and a definite efflux followed at 30 min. The results were indicative of cooperative effects during Se transport. Kinetic analyses of the uptake of 75Se during time intervals of 15 and 30 min were carried out both in the whole mitochondria and the mitochondrial protein fractions. Graphical analyses using Lineweaver-Burk plot, Hill plot, log [v] vs log [A] and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for 75Se uptake were biphasic, suggesting the probable presence of two classes of binding sites. The number of high and low affinity binding sites were estimated to be around 4-7 and 26-30 nmol/mg protein, respectively. Studies with mitochondrial respiratory inhibitors indicated about 10-20% of the total 75Se uptake to be energy dependent. Inhibition of 75Se uptake by about 60-70% by sulfate and sulfite (5-25 microM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of 75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of 75Se uptake by mitochondria of V. radiata emerges from the present study.