MITOCHONDRIAL SE-75 UPTAKE AND REGULATION REVEALED BY KINETIC-ANALYSIS

Uptake of (Na2SeO3)-Se-75 by mitochondria of the larvae of the insect C. cephalonica reared at different dietary selenium (Se) levels reveal ed: 1. A proportional increase in the uptake with externally added (Na 2SeO3)-Se-75 in concentrations upto 25.32 mu M; and 2. At each added s elenite concentration, an increase up to 60 min, with linearity up to 15-30 min. A differential affinity for (Na2SeO3)-Se-75 was elicited in the mitochondrial protein fractions of different dietary Se groups an d correlated well with the pattern and the ratio of distribution of in corporated Se-75 in protein to nonprotein fractions. Kinetic studies o n Se-75 uptake by whole mitochondria negated passive diffusion of sele nite and revealed a trend of negative cooperativity, confirmed by Hill and Scatchard plots. Half saturation value was estimated to be approx 13 nmole Se/mg mitochondrial protein. Scatchard plot for Se-75 uptake was biphasic and the high affinity binding sites were estimated to be around 5 nmole/mg mitochondrial protein. Calculated dissociation cons tants revealed maximal affinity for Se-75 in the 1.5 ppm group (K-Se 0 .0034 nM) and minimal in the basal group (K-Se 0.007 nM). In the mitoc hondria of all the three dietary Se groups, the estimated low affinity sites amounted to be 15-19 nmole/mg mitochondrial protein. Inherent S e in the mitochondria of the high Se group positively enhanced the inc orporation of Se-75 in the mitochondrial protein fraction. About 20-30 % of the total uptake was indicated to be energy linked as revealed by studies with respiratory inhibitors. Addition of sulfite and sulfate (5-25 mu M) in the medium, inhibited Se-75 uptake by 35-55%, suggestiv e of the involvement of the dicarboxylate port. Thiol interactive Se-7 5 uptake was confirmed by the inhibition mediated by mersalyl and NEM up to 50-70%. The study revealed thiol-selenite interactions of metabo lic significance during selenite uptake.