Activation of liver succinate dehydrogenase in rats exposed to hypobaric conditions

1. On brief exposure of rats to hypobaric conditions, the activity of hepatic mitochondrial succinate dehydrogenase was raised from the basal state to a ;partially activated state'. This was further raised to ;fully activated state' by preincubation of mitochondria with succinate, as was the activity in mitochondria from normal rats. 2. On washing mitochondria with the homogenizing sucrose medium the activity excess obtained on preincubation with succinate was lost in mitochondria from both normal and treated rats. 3. The enzyme in the ;partially activated state' from animals exposed to hypobaric conditions was stable to the washing procedure but was labilized and reverted to a low basal state of activity on freezing and thawing of the isolated mitochondria. 4. The results suggest that activation of succinate dehydrogenase under hypobaric conditions represents a conformational change leading to a stable, partially activated, form of the enzyme system: this is the first evidence of physiological modulation of this rate-limiting step in the control of the rate of oxidation of succinate.     

Inhibition of membrane-bound succinate dehydrogenase by fluorescamine

Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.     

Inhibition of membrane-bound succinate dehydrogenase by disulfiram

The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.     

Inhibition of mammalian succinate dehydrogenase by carboxins.

Carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide), a systemic fungicide, is known to inhibit the oxidation of succinate selectively in a variety of fungi and bacteria. Except for one report, the action of carboxin and of structurally related oxathiin derivatives on mammalian succinate dehydrogenase have not been investigated, however. In the present study, the inhibition of succinate oxidation by a number of carboxin derivatives have been studied using inner membrane preparations, purified particulate preparations (Complex II), and soluble preparations from beef heart. The site of action of carboxins has been studied by using a variety of electron acceptors. It has been concluded that carboxins inhibit mammalian succinate dehydrogenase by reacting at the same site as thenoyltrifluoroacetone but are effective at far lower concentrations. The maximal extent of inhibition by carboxins varies with the type of catalytic assay used and, in general, parallels the extent of inactivation brought about by cyanide, as if both types of agents modified the environment of an iron-sulfur component in the enzyme, presumably the superoxidized (HiPIP) Fe-S cluster.     

Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate

NaHCO3 activated the folylpolyglutamate synthetase (FPGS) from rat liver and the human leukemia cell lines K562 and CCRF-CEM by 1.7- to 2.0-fold. Optimal activation was achieved by 10 mM NaHCO3 in all cases; NaCl, sodium formate, sodium acetate, NaN3, and Na2SO3 at 10 mM did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. Km and Vmax values for pteroyl substrates (aminopterin or methotrexate) and L-glutamate increased proportionally in the presence of NaHCO3; there was thus no apparent change in the catalytic efficiency (Vmax/Km) of the FPGS reaction with these substrates. However, NaHCO3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent Km while increasing the Vmax of the reaction. NaHCO3 also activated FPGS activity when folic acid, dihydrofolic acid and tetrahydrofolic acid were substrates. The relative distribution of products synthesized from methotrexate or tetrahydrofolate by FPGS was not altered by addition of NaHCO3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO3 (IC50 = 0.4 microM). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.     

Enzyme electrokinetics: energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.