دانلود رایگان مقاله حساسیت دیفرانسیل کمپلکس میتوکندری دوم به بازداری

Abstract

Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd ~ 10− 8 M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2 ± 6.0% and 56.4 ± 5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

4. Discussion

Earlier investigations found that in preparations of SDH of various degree of purity, the enzyme was in a relatively inactivated state. Various treatments of the enzyme (ATP, reduced ubiquinone, TCA cycle metabolites and bromide ions) prior to activity measurement were used to obtain a higher, constant levels of activity [4–6,42]. It was revealed that most of these treatments resulted in dissociation of OAA tightly bound in the active center and consequent activation of the SDH [4–6,42]. If measured without any attempt to activate the enzyme by removal of OAA, progressive increase of the rate of succinate-oxidase reaction during onset of the assay can be observed. This activation is due to a slow dissociation of competitive inhibitor OAA from the active centre of the enzyme during continuous assay. OAA is a classical competitive inhibitor, with an extremely low dissociation rate (0.02 min−1 ) [7,43]. The lengths of the lag-phase depend on temperature, concentration of enzyme and substrate, as well as isolation protocol. Therefore, initial rates of the enzymatic reaction without activation do not reflect the full activity of the enzyme and could lead to underestimation of its real activity. In addition, alteration in concentration of TCA cycle intermediates in different conditions (i.e. normoxia/ischemia, wild type/mutant, the presence/absence of pharmacological agents) could affect the ratio between free and OAA-bound complex II in the preparation and complicate result interpretation.