Uptake of (75Se) added in vitro was followed in mitochondria isolated from Trigonella foenum-graecum seedlings grown under different Se status (0.5-1.0 ppm) and with added mimosine (0.1 mM). Uptake of 75Se followed with added Na2 75SeO3 up to 20 μM in the medium was nonlinear in all the groups. Kinetic analyses of the uptake of 75Se for 1 min were carried out for ali the groups. The results indicated a cooperative effect during Se transport. Graphical analyses using the Hill plot and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Scatchard plots were biphasic, suggesting the probable presence of two classes of binding sites. The presence of succinate or ATP in the incubation medium inhibited 75Se uptake by 40%. Studies with mitochondrial respiratory inhibitors indicated the uptake to be energy independent. A decrease in the uptake of 75Se by 40% effected by HgCl2, N-ethyl maleimide, and iodoacetate confirmed the interaction of active thiols in the process. The present study confirms the controlled nature of 75Se uptake by plant mitochondria.

Kalyanaraman Lalitha

IODOACETIC ACID

MERCURY CHLORIDE

MIMOSINE

N ETHYLMALEIMIDE

selenium

article

MITOCHONDRIAL RESPIRATION

Mitochondrion

nonhuman

PHYTOCHEMISTRY

plant seed

transport kinetics

GERMINATION

Mitochondria

Plants

SELENIUM RADIOISOTOPES

Time Factors

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Kinetic analyses of mitochondrial 75selenium uptake in Trigonella foenum-graecum seedlings exposed to selenium and mimosine.

Biological Trace Element Research

During the growth of selenium (Se)-deficient seedlings of Vigna radiata, exposure to mimosine [2-amino-3-(3-hydroxy-4-oxo-1H-pyridin-1-yl)-propanoic acid], a nonprotein plant amino acid, effectively mitigated stress at 0.1 mM, as reflected in enhancement of growth and efficiency of mitochondrial functions. Since the changes in the seedlings elicited by exposure to mimosine were similar to those effected by Se at an optimal exposure level of 0.75 ppm (Sreekala et al., Biol Trace Elem Res 70:193-207, 1999), the uptake of Se and that of mimosine itself was individually studied in the respiring mitochondria of Se-deficient seedlings (-Se-stressed group) in comparison with those exposed to mimosine during growth at 0.1 mM (Mim 0.1 group). In both groups, the mitochondrial uptake of 75Se at 10 μM added Na2 75 SeO3, increased linearly up to 2 min, attaining steady-state levels thereafter. Uptake levels were 2.3-fold higher in the Mim 0.1 group than in the -Se-stressed group. Double-reciprocal plots of mitochondrial 75Se uptake against 2-20 μM Na2 75 SeO3 in the medium were nonlinear and negative cooperative effects during the uptake were confirmed by Scatchard plots, whereas Hill coefficients were 0.8 and 0.85 for the two groups. Mitochondrial uptake of mimosine, at added levels of 25 or 50 μM, increased linearly up to 1 min and decelerated thereafter. Initial uptake levels of mimosine at 1 min were higher by 6.5-fold at 25 μM and 4-fold at 50 μM in the Mim 0.1 group than those in the -Se-stressed group. Initial uptake levels with added mimosine up to 50 or 100 μM yielded nonlinear double-reciprocal plots; and kinetic analyses at 5 to 50 μM revealed the prevalence of positive cooperativity in the -Se-stressed group and negative cooperativity in the Mim 0.1 group. Involvement of active thiol groups in the uptake of both Se and mimosine were indicated by inhibition studies. Evidence presented for mimosine mediated increase in mitochondrial Se uptake and cooperative interactions thereof underscores the metabolic significance of mimosine. © Humana Press Inc. 2007.

Mimosine mitigates oxidative stress in selenium deficient seedlings of Vigna radiata. Part II: Mitochondrial uptake of 75selenium and mimosine

Mitochondria from Trigonella foenum-graecum seedlings grown independently in the presence of either selenium (0.75 ppm) or mimosine (0.1 mM) exhibited respiration-stimulated energy-dependent uptake of Ca2+. Uptake studies were carried out independently at a series of Ca2+ concentrations at two different levels: (1) 1-20 μM and (2) 25-1500 μM. Levels of uptake were 50-100% higher in the mitochondria of seedlings of both the Se and mimosine groups. Detailed kinetic analyses revealed negative cooperative effects operative during uptake of Ca2+ at 25-1500 μM given in the medium. Hill coefficients for Ca2+ uptake by the mitochondria of different groups remained unchanged (nH, 0.75). Biphasic Scatchard plots were concave upward, suggestive of two classes of binding sites. High-affinity binding sites were estimated to be 16 nmol/mg protein with dissociation constant (KCa) of 2.5 × 109 L/mol. In contrast, graphical analyses of the uptake of Ca2+ in the range 1-20 μM in the medium revealed cooperative effects of positive nature. The present study demonstrates mixed cooperative effects during Ca2+ uptake by mitochondria from seedlings of T. foenum-graecum.

Uptake of 45Ca by mitochondria of Trigonella foenum-graecum as influenced by selenium and mimosine - Detailed kinetic analyses

Oxidative stress during selenium (Se) deficiency in the seedlings of Trigonella foenum-graecum grown for 72 h was investigated and the response to supplemented levels of Se (0.5-1 ppm) and mimosine (0.05-1 mM) was evaluated. Beneficial effects of Se was maximal at 0.75 ppm. Mimosine, a toxic amino acid, was also found to be beneficial to the growth of the seedlings exposed up to 0.2 mM. When compared to the stressed seedlings, mitochondrial oxygen uptake from seedlings of Se (0.75 ppm) group and mimosine (0.2 mM) group exhibited threefold enhancement in state 3 respiration rate and a controlled state 4 rate, with respiratory control ratios of 5-8. Upon supplementation at the optimal levels, superoxide dismutase (SOD) activities were enhanced fourfold with Se and eightfold with mimosine in the mitochondria. The soluble activity in mimosine groups increased twofold, but only by 75% in Se groups. Peroxidase activity registered a significant increase by threefold in mitochondria and fourfold in soluble fraction in both Se and mimosine groups. Exposure to Se or mimosine exhibited a differential response in the mitochondrial catalase and ascorbate peroxidase (Asc-Px) activities. In the Se groups, both catalase and Asc-Px in mitochondria decreased by 50-60%, which was contrasted by 60%, increase in Asc-Px activity and 40% in catalase activity in mimosine groups. Supplementation with either Se or mimosine evoked similar responses of increases with respect to soluble catalase by twofold to threefold and Asc-Px by 90%. The results of the present study reveal (1) the prevalence of oxidative stress in T. foenum-graecum during Se deficiency, (2) enhanced mitochondrial functional efficiency mediated by Se and mimosine independently, and (3) an antioxidative role for mimosine during Se deficiency. The study demonstrates for the first time that mimosine, a naturally occurring toxic amino acid, could be a beneficial growth factor in concentrations between 0.1 and 0.2 mM.

Oxidative stress during selenium deficiency in seedlings of Trigonella foenum-graecum and mitigation by mimosine: Part I. Hydroperoxide metabolism

Adaptive alterations in glutathione (GSH) metabolism were studied during oxidative stress induced by selenium (Se) deficiency in germinating seedlings of Trigonella foenum-graecum grown for 72 h and the response to supplementation individually of Se or mimosine was explored. Growth enhancement with improved mitochondrial efficiency was elicited by supplementation of Se at 0.5-0.75 ppm or mimosine at 0.1-0.2 mM. Total thiol and protein levels of mitochondrial and soluble fractions, in general, did not vary significantly with supplementation of either Se or mimosine except that the mitochondrial protein levels in mimosine groups (0.1-0.2 mM) decreased by 20-30%. Mitochondrial glutathione peroxidase (GSH-Px) increased by twofold in activity toward H2O2, cumene hydroperoxide (CHP), and t-butyl hydroperoxide (tBHP) in Se groups, and by 50-60% increase toward H2O2 and CHP but by a twofold enhancement in enzyme activity with tBHP in mimosine groups. Soluble GSH-Px activity increased by 30-40% only in mimosine groups and remained unaltered in Se groups. Glutathione S-transferase activity (GST) in the soluble fraction of both Se and mimosine groups increased dramatically by fivefold to sixfold. Distinct differences were noted in the response of the stressed seedlings toward exposure to Se or mimosine and included a decline in glutathione reductase (GR) activity by 50-60% in both mitochondria and soluble fractions of Se groups and an increase in GR activity of the mitochondria by twofold and of the soluble enzyme activity by 30% in the mimosine groups. Mimosine exposure resulted in a dose-dependent decrease in the γ-glutamy transpeptidase levels, but, in contrast, a significant enhancement by 50% was noted in the Se group at 0.75 ppm. The results including the differential response of GR activity to Se or mimosine supplementation are reflective of an effective reductive environment in Se group, and increased turnover of GSH in the presence of mimosine.

Oxidative stress during selenium deficiency in seedlings of Trigonella foenum-graecum and mitigation by mimosine: Part II. Glutathione metabolism

Selenium (Se) deficiency in the experimental models, Coturnix coturnix japonica and Corcyra cephalonica, resulted in impaired mitochondrial substrate oxidations and lowered thiol levels. Studies with respiratory inhibitors confirmed reduced mitochondrial electron transport enzyme activities, especially at cytochrome c oxidase (COX), the terminal segment. Enhanced mitochondrial lipid peroxidation in Se deficiency was more pronounced in the heart tissue of the quail compared to other tissues. Glutathione peroxidase (GSH-Px) activity toward H2O2 and cumene hydroperoxide were generally low in the insect muscle tissue and activity toward H2O2 was maximal in the quail heart mitochondria that was not very sensitive to Se status. Lowered COX activity in Se deficiency was more directly correlated with the increased level of lipid peroxidation than with the GSH-Px activity measured, suggestive of Se mediated protective mechanisms independent of GSH-Px. Electron microscopic observations revealed structural changes such as loss of cristae with proliferative and degenerative changes of the mitochondria in Se deficiency. Involvement of Se in maintaining structure and functional efficiency of mitochondria is evident from the present study.

Evidence for altered structure and impaired mitochondrial electron transport function in selenium deficiency

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 of75Se. Uptake of Na275SeO3, 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 Na275SeO3, in concentrations up to 25 γM. The uptake of75Se, 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 of75Se 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 during75Se uptake. Hill coefficient (nH) values were estimated to be around 0.7-0.8. Scatchard plots for75Se 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 total75Se uptake to be energy dependent. Inhibition of75Se uptake by about 60-70% by sulfate and sulfite (5-25 γM) implies the involvement of dicarboxylate port in Se transport. A decrease in the uptake of75Se by 40-60% effected by CdCl2, HgCl2, mersalyl, and NEM confirmed the interaction of thiols in the process. Evidence for the regulatory nature of75Se uptake by mitochondria of V. radiata emerges from the present study. © 1995 Humana Press Inc.

Kinetic analysis of75selenium uptake by mitochondria of germinating vigna radiata of different selenium status

Uptake of Na275SeO3 by mitochondria of the larvae of the insect C. cephalonica reared at different dietary selenium (Se) levels revealed: 1. A proportional increase in the uptake with externally added Na275SeO3 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 Na275SeO3 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 (KSe 0.0034 n M) and minimal in the basal group (KSe 0.007 n M). 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. © 1995 Humana Press Inc.

Journal	Biological Trace Element Research
ISSN	01634984
Open Access	No