**5.5. Dual regulatory effect of ISC on SDH activity in the area of physiological regulation. GTP-recoupling but not oxaloacetate (OAA)-inhibition regulates SUC oxidation under physiological conditions**

ISC is a natural efficient intramitochondrial antioxidant [54]. Reducing equivalents produced by ISC NAD(P)-dependent dehydrogenases protect the most sensitive enzymes aconitase and KDH from oxidation and increase the stability of mitochondria to oxidative stress, which accompanies any stress**.** It was shown long ago that ISC abolishes OAA inhibition of SDH in aged mitochondria or under severe stress [50, 55]. This effect is due to OAA elimination by reduction into malate because of increase in the reduction of the respiratory chain. A similar effect is usually attained by the addition of glutamate, which eliminates OAA via transamination. As distinct from glutamate, the application of ISC for the elimination of SDH inhibition was nearly forgotten. This may be due to the fact that its fully active natural isomer is commercially hardly available, while the available chemical racemate is less active. Fortunately, due to the collaboration with microbiologists manufacturing natural ISC, we had an opportunity to investigate this substance [56].

Our investigation of the influence of natural ISC on SDH activity during PES under more physiological conditions of the CBCh method discovered an unknown effect of this substrate. In spite of acceleration of inhibited SUC oxidation, ISC strongly suppresses hyperactivated SDH. This effect is shown in Figure 7. It is clearly seen that ISC addition suppresses greatly SDH activity in the hyperactivated state, 30, 60, 120 min of PES.

oxygen [53].

activities are at the lowest level.

Together with this traditional effect of MAL, we revealed the opposite action of MAL, namely, an increase in NBT reduction. It was most pronounced in the subgroup Activation with Inhibition, which was distinguished according to a decreased SDH activity relative to the Activation subgroup. One cause of the inhibition (example 20) could be an elder age and initial hypertension demanding the intake small doses of adrenoblockers, which also inhibits SDH. In this case, an increase in NBT reduction by MAL is the highest. This effect is also well pronounced relatively to ES in two other examples in this subgroup. This unknown effect of MAL on NBT reduction is also observed in Deep Quiescence, in which all

Thus, MAL manifests the ability to increase NBT reduction in case of low SDH activity, in

In our opinion, the increase in NBT reduction by MAL is a marker of ROS formation in mitochondria. As shown in Figure 7, ROS formation sharply rises at the beginning of stress. During stress, the rise of ROS formation declines as evidenced by our measurements with MAL. This agrees with other results [52]. As shown in the last example in Figure 7, ROS rise again by the exhausting stage. The biochemical mechanism of this effect of MAL is in deep inhibition of SDH, which leads to a fall of coenzyme Q reduction from full reduction to free radical form. Free radical form of Q produces superoxide in interaction with surrounding

**5.5. Dual regulatory effect of ISC on SDH activity in the area of physiological regulation. GTP-recoupling but not oxaloacetate (OAA)-inhibition regulates** 

ISC is a natural efficient intramitochondrial antioxidant [54]. Reducing equivalents produced by ISC NAD(P)-dependent dehydrogenases protect the most sensitive enzymes aconitase and KDH from oxidation and increase the stability of mitochondria to oxidative stress, which accompanies any stress**.** It was shown long ago that ISC abolishes OAA inhibition of SDH in aged mitochondria or under severe stress [50, 55]. This effect is due to OAA elimination by reduction into malate because of increase in the reduction of the respiratory chain. A similar effect is usually attained by the addition of glutamate, which eliminates OAA via transamination. As distinct from glutamate, the application of ISC for the elimination of SDH inhibition was nearly forgotten. This may be due to the fact that its fully active natural isomer is commercially hardly available, while the available chemical racemate is less active. Fortunately, due to the collaboration with microbiologists

manufacturing natural ISC, we had an opportunity to investigate this substance [56].

Our investigation of the influence of natural ISC on SDH activity during PES under more physiological conditions of the CBCh method discovered an unknown effect of this substrate. In spite of acceleration of inhibited SUC oxidation, ISC strongly suppresses hyperactivated SDH. This effect is shown in Figure 7. It is clearly seen that ISC addition suppresses greatly SDH activity in the hyperactivated state, 30, 60, 120 min of PES.

**SUC oxidation under physiological conditions** 

contrast to its traditional inhibition of respiration is great when SDH activity is high.

However, ISC preserves the ability to stimulate inhibited SDH under 180- min PES. Besides, we have found that ISC does not influence the SDH activity in the Quiescent State. The two opposite effects of ISL: an increase and a decrease in SDH activity are easily explained by the single antioxidant mechanism. The increase in NBT reduction by MAL at the beginning of PES shows the development of oxidative stress. This agrees with other observations. The level of oxidative stress is regulated by well-balanced interactions of several processes occurring in mitochondria. Among them are initial uncoupling and its secondary recoupling by GTP [52].

Recoupling effect of GTP can explain our observation that preserving the KDH, which is a real source of GTP-formed in substrate-level phosphorylation, protects SDH from hyperactivation followed by inhibition.

NBT reduction is expressed as a total area of formazan granules in 30 lymphocytes.

Owing to a great number of objects calculated (200-800) the presented data are statistically significant at the level of 0.001 (99.99%)

In this metabolic environment, protection of KDH from oxidation by ISC allows one to support GTP generation and to diminish the uncoupling that was a cause of initial SDH hyperactivation. This explanation agrees well with our repeated observations that the preservation of high KDH activity simultaneously with activation of SDH is typical of activity within physiological adaptation area. The "scissors" between SDH rise and KDH fall are observed in severe stress or pathology. Therefore, three different effects of ISL on SDH activity: suppression of hyperactivation, activation of inhibition, and absence of influence in norm, allow one to differentiate clearly functional states of SDH. The area of

SDH regulation involving KDH-dependent GTP – recoupling - is not known in biochemical investigations because SDH is initially hyperactivated under the experimental conditions. The hyperactivation of SDH elevates the risk of inhibition by OAA due to the increase in its formation. Indeed, OAA inhibition is often observed, when SUC oxidation is accelerated, for example, by uncoupling. Therefore, OAA inhibition is considered to be the only mechanism of SDH control. However, this is true for in vitro conditions in biochemical experiments in isolated mitochondria. The preservation of the area more close to physiological conditions in the CBCh method opens Terra Incognita of unknown regulation of the SDH activity by GTP-recoupling instead of usual OAA inhibition.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 253

**Figure 8.** Responses of ES and SDH to activation by administration of adrenaline or succinate measured

A - calm rabbit, B - restless rabbit. SDH activity is expressed as a mean area of formazan granules in 30 cells. The mean of three measurements in different days for each rabbit are presented, M ± SEM. \* - р< 0.05, \*\* - p<0.01, comparing to the respective initial state, # - p<0.05, comparing to the calm rabbit.

As one of the models of domination of these regulation types we studied individual

One rabbit was calm during the experiment; another rabbit escaped and was anxious and restless even when it was not exposed to treatment. They represented characteristic types of a cholinergic and adrenergic, respectively. Figure 8 demonstrates significant differences

The reserve of ES in the calm rabbit is much greater than in the restless. After the injection of ADR, the level of ES in the calm rabbit decreases; however, this level is higher than in the restless animal under the influence of ADR. In the intact restless animal, the level of ES is 15 times lower than that of the intact calm rabbit and 5 times lower than in the calm animal after the injection of ADR. This indicates an enhanced expenditure of substrates in the

The SDH activity in the calm rabbit is low, and the injections of ADR or SUC strongly stimulates it. The stimulation of SUC is somewhat stronger, which increases the risk of the transition from hyperactivation to inhibition. By contrast to ADR, the injection of SUC also

between the calm and restless rabbits in the level of ES and in the SDH activity.

in lymphocytes of rabbits with different behavior.

restless animal.

increases the level of ES.

differences of CBCh patterns in rabbits with different behaviour.

The last but not least, it is worth mentioning that the mutual reciprocal regulation of SDH and KDH activity is further evidence of ADR and ACH regulation in mitochondria. In essence, it corresponds to the reciprocal influence of ADR and ACH on physiological functions: stimulation, and restriction with restoration, respectively.

Before continuation, let us put in a brief insertion to memorize the better approach of CBCh determination of SDH activity to mitochondrial processes compared to pure enzymatic measurements.
