Monoamine oxidase and mitochondrial respiration

Mitochondrial defects encompassing complexes I-IV of the electron transport chain characterize a relatively large number of neurodegenerative diseases. The relationships between mitochondrial lesions and recently described genetic alterations have not yet been defined. We describe a general mechanism whereby the enzymatic metabolism of neurotransmitters by monoamine oxidase (MAO) damages mitochondria, altering their protein thiol status and suppressing respiration. In these experiments, incubation of rat brain mitochondria with tyramine (a mixed MAO-A/MAO-B substrate) for 15 min at 27 degrees C suppressed state 3 respiration by 32.8% and state 5 respiration by 40.1%. These changes were accompanied by a 10-fold rise in protein-glutathione mixed disulfides. Direct comparison of effects on respiration and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction during electron flow gave similar results. It is suggested that certain mitochondrial lesions may derive from the natural turnover of monoamine neurotransmitters in susceptible individuals.     

Visualization of mitochondrial respiratory function using cytochrome c oxidase/succinate dehydrogenase (COX/SDH) double-labeling histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations (1,2). The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process (3). A wealth of evidence has been compiled in support of this theory (1,4), an example being the mtDNA mutator mouse (5); however, the precise role of mtDNA damage in aging is not entirely understood (6,7). Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity (2). Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination (8,9). Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis (10-12). The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity (2,12-14). Although COX and SDH activities can be investigated individually, the sequential double-labeling method (15,16) has proved to be advantageous in locating cells with mitochondrial dysfunction (12,17-21). Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time (9,22,23). 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor (22). In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells (22). Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product (9,24). Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues (9). Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity (9,22). Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products (9,25). Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.     

Inner mitochondrial membranes bound to concanavalin A-sepharose display succinate dehydrogenase, ATPase, and cytochrome oxidase activity

A fraction (15-20% of the total protein) of a preparation of bovine submitochondrial particles (SMPs) binds to concanavalin A-sepharose. The bound membranes displayed succinate dehydrogenase, cytochrome oxidase, and ATPase activity, which, as in SMPs, were inhibited by malonate, cyanide, and oligomycin, respectively. These results indicate that the bound membranes are inner mitochondrial membranes and that they contain a glycoprotein which was recognized by concanavalin A. It was possible to repeatedly perform the three enzyme assays, one after the other, in the same gel with the bound membranes. Long-term stability tests (22 days) showed that cytochrome oxidase was much more stable in the membranes bound to the gel than in SMPs, while the ATPase activity decayed at a similar rate in the two conditions. Thus, inner mitochondrial membranes bound to ConA-Sepharose appear to be a potentially interesting model for the study of immobilized multienzymatic complexes.     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Two sites of ubisemiquinone binding in mitochondrial succinate oxidase

The inhibiting effects of several phenol compounds on electron transfer in the respiratory chain of submitochondrial particles were studied. It was shown that the terminal part of the succinate dehydrogenase complex contains a site, which specifically binds the negatively charged phenols (e. g. pentachlorophenol, 2,4-dibromophenol, 2-methoxy-4,6-dibromophenol). The efficiency of the inhibitor anion binding by this site is increased 12-fold after introduction of a methoxy-group into the o-position of 2,4-dibromophenol. Since this site binds both methoxy- and negatively charged phenol groups, it can also act as a possible site of ubisemiquinone interaction with succinate dehydrogenase. Based on the structural similiarity of the b-c1 complex inhibitors, e. g. antimycin, o-hydroxybenzoic acid amides, 2-hydroxy-3-alkyl-1,4-quinones, and ubisemiquinone, an assumption has been made on possible ubisemiquinone binding between cytochromes b and c1. A structural analysis of phenols inhibiting succinate dehydrogenase and the b-c1 complex revealed that in the region between cytochromes b and c1 there act only the inhimitors which contain: a) a negatively charged phenol group; b) a group comprising a heteroatom with an undepleted electron pair and which can act as a ligand, and c) a hydrophobic residue.     

Modulation of mitochondrial succinate dehydrogenase activity,mechanism and function

Summary The mitochondrial succinate dehydrogenase (E.C. 1.3.3.99) is subjected to apparently complicated regulatory mechanism. Yet, systematic analysis of the mechanism reveals the simplicity of the control. There are two stable forms of the enzyme; the non-active form stabilized as 1:1 complex with oxaloacetate and the active form stabilized by binding of activating ligands. This model quantitatively describes either the equilibrium level of active enzyme or the kinetics of activation-deactivation, in the presence of various concentrations of opposing effectors. The site where the regulatory ligands interact with the enzyme is not the substrate bonding site. The marked differences of dissociation constants of the same ligand from the two sites clearly distinguish between them.This model is fully developed for simple cases where the activating ligands are dicarboxylic acids or monovalent anions. On the other hand with activators such as ATP or CoQH₂, quantitation is still not at hand. This stems from the difficulties in maintaining determined, measurable, concentrations of the ligand in equilibrium with the membranal enzyme.While in active form the histidyl flavin moity of the enzyme is reduced by physiological substrate (succinate; CoQH₂). The non-active form is not reduced by these compounds, only strong reductants with low redox potential reduce the non-active enzyme. It is suggested that deactivation is a simple modulation of the redox potential of the flavin form E 0 mV in the active enzyme to E < –190 mV. The switch from one state to another might be achieved by distortion of the planar form of oxidized flavin to the bend configuration of the reduced flavin. Thus, in the active enzyme such distortion will destabilize the oxidized state of the flavin, shifting the redox potential to the higher value. The binding of oxaloacetate to the regulatory sites releases the distorting forces by relaxing the conformation of the enzyme. Consequently, the flavin assumes its planar form with the low redox potential. This assumption is supported by the spectral shifts of the flavin associated with the activation deactivation transition.The suicidal oxidation of malate to oxaloacetate, carried by the succinate dehydrogenase, plays an important role in modulating the enzyme activity in the mitochondria. This mechanism might supply oxaloacetate for deactivation in spite of the negligible concentration of free oxaloacetate in the matrix. The oxidation of malate by the enzyme is controlled by the redox potential at the immediate vicinity of the enzyme, and is imposed by the redox level of the membranal quinone.Finally, the modulation of succinate dehydrogenase activity is closely associated with regulation of NADH oxidation through the mutual inhibition between oxidases (Gutman, M. in Bioenergetics of Membranes, L. Packer et al., ed. Elsevier 1977, p. 165). The consequence of these interactions is the selection for the main electron donnor for the respiratory chain, during mixed substrate respiration, according to the metabolic demands from the mitochondria.Abbreviations SDH succinate dehydrogenase (succinate: acceptor oxidoreductase (E.C. 1.3.99.1)); - OAA oxaloacetate - Act activator - E_A, E_A active and non active forms of the enzyme, respectively - K'eq apparent equilibrium constant - K'd apparent dissociation constant - K_Act, K_OAA dissociation constant of the respective ligand from the enzyme - K'a, k'd the apparent rate constants of activation and deactivation, respectively - ka, kd the true rate constant of activation and deactivation respectively - ETP, ETP_II non phosphorylating and phosphorylating submitochondrial particles - PMS phenazine methosulfate - DCIP dichlorophenol indophenol - CoQ ubiquinone - TIFA Thenotriflouvoacetone - NEM N methyl Maleimide     

Tumor necrosis factor induces activation of mitochondrial succinate dehydrogenase

We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164.     

Proapoptotic action of alpha-tocopheryl succinate in rat thymocytes is conditioned by the inhibition of mitochondrial succinate dehydrogenase

It has been established that alpha-tocopheryl succinate in concenrations 10-100 microM inhibits in a dose-dependent manner the viability of primary culture rats thymocytes and causes the DNA internucleosomal degradation that testifies to apoptotic way of thymocytes destruction. These effects were accompanied by an enhanced production of intracellular superoxide. This is the first report demonstrating that apoptosis induced by alpha-tocopheryl succinate was accompanied by a dose-dependent inhibition of mitochondrial succinate dehydrogenase. Known apoptosis inducers--actinomicin D, staurosporin and hydrogen peroxide decreased a cell survival but neither induced any significant changes in succinate dehydrogenase activity which means that this effect is characteristic only of alpha-tocopheryl succinate and seems to be an important event triggering the apoptotic response by it. It was supposed that alpha-tocopheryl succinate might appear as a pseudosubstrate for mitochondrial succinate dehydrogenase leading to its inhibition, dysfunction of the mitochondrial electron transport chain, generation of reactive oxygen species and iduction of apoptosis.     

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.     

Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity

Poliovirus infection of COS-1 and T47D cells caused a rapid decrease in total cell respiration, and this was attributed to an inhibition of mitochondrial respiration. The stimulation of mitochondrial respiration by pyruvate plus malate or succinate was impaired in saponin-permeabilised cells. However, this inhibition could be overcome by the addition of N,N,N',N'-tetramethyl-1, 4-phenylenediamine and ascorbate. The activity of succinate dehydrogenase was impaired in parallel with the inhibition of mitochondrial respiration during poliovirus infection. This shows that mitochondrial function is profoundly altered during poliovirus infection and that this occurs primarily through inhibition of electron flow at complex II of the mitochondrial respiratory chain.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.     

Cell-free synthesis of succinate dehydrogenase and mitochondrial adenosine triphosphatase of sweet potato

Polyadenylated mRNA was isolated from aged slices of sweet potato root tissue and translated in a wheat germ cell-free system. The synthesis of apoprotein of the flavoprotein subunit of succinate dehydrogenase and two of the subunits of mitochondrial adenosine triphosphatase were detected by indirect immunoprecipitation. The molecular weights of the immunologically identified products were 3,000 and 8,000-9,000 daltons larger than the mature flavoprotein subunit of succinate dehydrogenase and the mature subunits of adenosine triphosphatase, respectively.     

Mapping of mitochondrial metabolic competence by cytochrome oxidase and succinic dehydrogenase cytochemistry.

To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/microm(3) of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:1191-1192, 2001)