Rotenone-insensitive NADH Dehydrogenase Research Articles

The nuclear origin of the non-phosphorylating NADH dehydrogenases of plant mitochondria

The oxidation of matrix and cytosolic NADH by isolated beetroot and wheat leaf mitochondria was investigated to determine whether the rotenone-insensitive NADH dehydrogenases of plant mitochondria were the products of nuclear or mitochondrial genes. After aging beetroot tissue (slicing and incubating in a CaSO 4 solution), the induction of the level of matrix NADH oxidation in the presence of rotenone was greatly reduced in mitochondria isolated from tissue treated with cycloheximide, a nuclear protein synthesis inhibitor. This was also true for the oxidation of cytosolic NADH. Mitochondria isolated from chloramphenicol-treated tissue exhibited greatly increased levels of both matrix and external rotenone-insensitive NADH oxidation when compared to the increase due to the aging process alone. This increase was not accompanied by an increase in matrix NAD-linked substrate dehydrogenases such as malic enzyme nor intra-mitochondrial NAD levels. Possible explanations for this increase in rotenone-insensitive NADH oxidation are discussed. Based on these results we have concluded that the matrix facing rotenone-insensitive NADH dehydrogenase of plant mitochondria is encoded by a nuclear gene and synthesis of the protein occurs in the cytosol.

Specific interaction of cytokinins and their analogs with rotenone-sensitive internal NADH dehydrogenase in potato tuber mitochondria.

Effects of cytokinins were studied on rotenone-sensitive NADH dehydrogenase in mitochondria from fresh potato tubers (Solanum tuberosum), in consideration of the operation of external and rotenone-insensitive internal NADH dehydrogenases that has not been fully accounted for in previous studies. In submitochondrial particles (smp), zeatin was only weakly active, and zeatin riboside (ZR) was inactive. Inhibition rates at 400 microns of isopentenyladenine (iP) and isopentenyladenosine (iPA) were 45% and 30%, respectively, and that of BA (BA) was 64%. In intact mitochondria, the inhibition by iP and BA significantly increased, I50 being 50 and 250 microM, respectively, but that by zeatin and iPA decreased. A structure-activity study showed that hydrophobic and steric factors are important for the activity. Cytokinins inhibited the electron flow via natural quinone more strongly than that via synthetic quinone. These results suggest that among the cytokinins the species that can regulate the electron transport is iP rather than its riboside or zeatin.

Ubiquinone-1 Induces External Deamino-NADH Oxidation in Potato Tuber Mitochondria.

The addition of ubiquinone-1 (UQ-1) induced Ca2+-independent oxidation of deamino-NADH and NADH by intact potato (Solanum tuberosum L. cv Bintje) tuber mitochondria. The induced oxidation was coupled to the generation of a membrane potential. Measurements of NAD+-malate dehydrogenase activity indicated that the permeability of the inner mitochondrial membrane to NADH and deamino-NADH was not altered by the addition of UQ-1. We conclude that UQ-1-induced external deamino-NADH oxidation is due to a change in specificity of the external rotenone-insensitive NADH dehydrogenase. The addition of UQ-1 also induced rotenone-insensitive oxidation of deamino-NADH by inside-out submitochondrial particles, but whether this was due to a change in the specificity of the internal rotenone-insensitive NAD(P)H dehydrogenase or to a bypass in complex I could not be determined.

Purification and Characterization of a 43-kDa Rotenone-insensitive NADH Dehydrogenase from Plant Mitochondria

A 43-kDa NAD(P)H dehydrogenase was purified from red beetroot mitochondria. An antibody against this dehydrogenase was used in conjunction with the membrane-impermeable protein cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) to localize the dehydrogenase on the matrix side of the inner membrane. Immunoblotting showed that the dehydrogenase was found in mitochondria isolated from several plant species but not from rat livers. Antibodies against the purified dehydrogenase partially inhibited rotenoneinsensitive internal NADH oxidation by inside-out submitochondrial particles. The level of rotenone-insensitive respiration with NAD-linked substrates correlated with the amount of 43-kDa NAD(P)H dehydrogenase present in mitochondria isolated from different soybean tissues. Based on these results, we conclude that the 43-kDa NAD(P)H dehydrogenase is responsible for rotenone-insensitive internal NADH oxidation in plant mitochondria.

Oxidation of Cytosolic NADH via Complex I of Heart Mitochondria

The exogenous NADH dehydrogenase of heart mitochondria is increasingly reported to mediate cardiomyopathies following adriamycin treatment or reperfusion of ischemic hearts. A great number of studies on the biochemistry and pathobiology exists which indirectly support the existence of this dehydrogenase. Our studies exclude both the rotenone-insensitive NADH dehydrogenase of the outer membrane and the endogenous NADH dehydrogenase of damaged mitochondria as being responsible for external NADH consumption. Reducing equivalents from external NADH were demonstrated to enter complex I of the respiratory chain from the cytosolic phase. Our data support earlier reports on the physical association of the exogenous NADH dehydrogenase with the inner mitochondrial membrane excluding oxidation of external NADH via an enzyme of artefactual origin.

Nadh: Ubiquinone oxidoreductase in obligate aerobic yeasts

The strictly aerobic yeasts Candida pinus, Cryptococcus albidus, Rhodotorula minuta, Rhodotorula mucilaginosa and Trichosporon beigelii possess mitochondrial NADH dehydrogenases with significant features of the NADH:ubiquinone oxidoreductase (complex I). These species show in all growth phases and under standard cultivation conditions, NADH dehydrogenases of approximately 700 kDa, which are sensitive to rotenone, a specific inhibitor of this complex. Identical results were obtained with the weakly fermenting C. pinus. The facultatively fermenting yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus do not possess the 700 kDa-complex and are insensitive to rotenone. In S. cerevisiae, a rotenone-insensitive NADH dehydrogenase of about 500-600 kDa is detected only in stationary phase cells. As in Neurospora crassa, upon incubation of the obligately aerobic yeast R. mucilaginosa with chloramphenicol, an intermediate NADH dehydrogenase of approximately 350 kDa was formed, which was insensitive to rotenone.

Partial Purification and Characterization of Complex I, NADH:Ubiquinone Reductase, from the Inner Membrane of Beetroot Mitochondria

A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH-ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone-sensitive NADH oxidation by beetroot submitochondrial particles.

Reactivation of the NADH-Q1 reductase activity of the isolated rotenone-insensitive NADH dehydrogenase of Arum maculatum mitochondria. Activation of quinone reductase activity by a hydrophobic component extractable from Arum mitochondrial membranes

The rotenone-insensitive NADH dehydrogenase of Arum maculatum mitochondria is inactivated during the process of purification. The ubiquinone reductase activity is inactivated while the dichlorophenolindophenol (DCIP) reductase activity is not. The former activity can be largely restored by an ether-extractable component(s) from Arum mitochondrial membranes which co-purifies with the enzyme. Thin-layer chromatography showed that the component responsible for maintaining ubiquinone reductase activity co-purifies with the enzyme and is not ubiquinone-10 or phosphotidyl choline. Up to 80% of the original activity was restored by the ether extract. These observations show that the rotenone-insensitive NADH dehydrogenase, inactivated during purification can be reactivated after the isolation process.

Properties of a soluble rotenone-insensitive NADH dehydrogenase released from Arum maculatum mitochondrial membranes by sonication

Sonication of Arum maculatum mitochondrial membranes resulted in the release of a soluble fraction which was enriched in a NADH: ubiquinone-1 reductase activity. The enzyme reduced ubiquinone and duroquinone at rapid rates. Its insensitivity to rotenone and inhibition by flavone indicate that the activity is due to the external NADH dehydrogenase. The enzyme is specific for NADH above pH 6.8 and has a K m for NADH of 27 μM at pH 7.2 with DCIP as acceptor. The solubilized NADH dehydrogenase occurred in solution in aggregated forms which could be detected by activity staining of the enzyme on polyacrylamide gels. Second-dimension electrophoresis of the various forms in the presence of sodium dodecyl sulphate revealed numerous polypeptides, of which two, of molecular weights 39 200 and 33 000, were present in all bands of activity. On the basis of its absorption spectrum, the enzyme appears to contain NADH-reducible flavin. No ESR-detectable iron-sulphur centres were detected other than those attributable to a contamination by succinate dehydrogenase.

Purification and characterization of the rotenone-insensitive NADH dehydrogenase of mitochondria from Arum maculatum.

The non-ionic detergent lauryl dimethylamine N-oxide (LDAO) has been used to extract the NADH dehydrogenases of Arum maculatum mitochondria. Affinity chromatography on 5'-ADP-Sepharose 4B was used to separate the rotenone-sensitive (complex I) NADH dehydrogenase from the rotenone-insensitive NADH dehydrogenase. An 18-fold purification of the rotenone-insensitive NADH dehydrogenase was achieved. The enzyme is specific for NADH with optimal activity around pH 7.2. The apparent Km for NADH is 28 microM, with dichloroindophenol as acceptor at pH 7.2. The rotenone-insensitive NADH dehydrogenase appears to be a flavoprotein and no iron-sulphur centres were detected by electron spin resonance spectroscopy.

Changes in the electron transport chain of pea leaf mitochondria metabolizing malate

Pea leaf mitochondria showed complex kinetics for malate metabolism. O 2 uptake increased as malate concentration increased from 0 to 10 m m, reached a plateau between 10 and 20 m m malate, and then increased again up to 40 m m malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O 2 uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O 2 uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O 2 uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing K m values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 m m malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O 2 through the alternative oxidase. Although this coincided with the second phase of malate-dependent O 2 uptake it was not required for this phase to be seen.

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with alpha-ketoglutarate and malate as substrates. Whereas alpha-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD(+), glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

MALATE OXIDATION IN PLANT MITOCHONDRIA PROCEEDS THROUGH THE ACTIVITIES OF TWO ENZYMES: a malate dehydrogenase and a NAD(+)-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD(+) stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD(+) are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD(+)/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD(+)/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.

Malic enzyme activity and cyanide-insensitive electron transport in plant mitochondria

In the presence of exogenous NAD +, malate oxidation by cauliflower mitochondria takes place essentially via an electron transport pathway that is insensitive to rotenone, antimycin and cyanide but is strongly sensitive to salicyl hydroxamic acid. It bypasses all phosphorylation sites. NAD + is reduced by an enzyme identified as malic enzyme (L-malate:NAD oxidoreductase (decarboxylating), EC 1.1.1.39). The NADH produced is reoxidized by an internal rotenone-insensitive NADH dehydrogenase that yields electrons directly to the cyanide-insensitive pathway.