Mitochondrial selenium-75 uptake and regulation revealed by kinetic analysis

Abstract

Uptake of Na₂ ⁷⁵SeO₃ by mitochondria of the larvae of the insectC. cephalonica reared at different dietary selenium (Se) levels revealed:

• 1. A proportional increase in the uptake with externally added Na₂ ⁷⁵SeO₃ in concentrations upto 25.32 μM; and

• 2. At each added selenite concentration, an increase up to 60 min, with linearity up to 15–30 min.

A differential affinity for Na₂ ⁷⁵SeO₃ was elicited in the mitochondrial protein fractions of different dietary Se groups and correlated well with the pattern and the ratio of distribution of incorporated⁷⁵Se in protein to nonprotein fractions. Kinetic studies on⁷⁵Se uptake by whole mitochondria negated passive diffusion of selenite 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 for⁷⁵Se uptake was biphasic and the high affinity binding sites were estimated to be around 5 nmole/mg mitochondrial protein. Calculated dissociation constants revealed maximal affinity for⁷⁵Se in the 1.5 ppm group (K_Se 0.0034 nM) and minimal in the basal group (K_Se 0.007 nM). In the mitochondria of all the three dietary Se groups, the estimated low affinity sites amounted to be 15–19 nmole/mg mitochondrial protein. Inherent Se in the mitochondria of the high Se group positively enhanced the incorporation of⁷⁵Se 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 μM) in the medium, inhibited⁷⁵Se uptake by 35–55%, suggestive of the involvement of the dicarboxylate port. Thiol interactive⁷⁵Se uptake was confirmed by the inhibition mediated by mersalyl and NEM up to 50–70%. The study revealed thiol-selenite interactions of metabolic significance during selenite uptake.