The effect of rotenone on respiration in pea cotyledon mitochondria

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of α-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.     

Regulation of malate oxidation in plant mitochondria. Response to rotenone and exogenous NAD+.

Exogenous NAD+ stimulated the rotenone-resistant oxidation of all the NAD+-linked tricarboxylic acid-cycle substrates in mitochondria from Jerusalem artichoke (Helianthus tuberosus L.) tubers. The stimulation was not removed by the addition of EGTA, which is known to inhibit the oxidation of exogenous NADH. It is therefore concluded that added NAD+ gains access to the matrix space and stimulates oxidation by the rotenone-resistant NADH dehydrogenase located on the matrix surface of the inner membrane. Added NAD+ stimulated the activity of malic enzyme and displaced the equilibrium of malate dehydrogenase; both observations are consistent with entry of NAD+ into the matrix space. Analysis of products of malate oxidation showed that rotenone-resistant oxygen uptake only occurred when the concentration of oxaloacetate was low and that of NADH was high. Thus it is proposed that the concentration of NADH regulates the activity of the two internal NADH dehydrogenases. Evidence is presented to suggest that the rotenone-resistant NADH dehydrogenase is engaged under conditions of high phosphorylation potential, which restricts electron flux through the rotenone-sensitive dehydrogenase (coupled to ATP synthesis).     

Malate oxidation, rotenone-resistance, and alternative path activity in plant mitochondria

The effect of cyanide and rotenone on malate (pH 6.8), malate plus glutamate (pH 7.8), citrate, α-ketoglutarate, and succinate oxidation by cauliflower (Brassica oleracea L.) bud, sweet potato (Ipomoea batatis L.) tuber, and spinach (Spinacia oleracea and Kalanchoë daigremontiana leaf mitochondria was investigated. Cyanide inhibited all substrates equally with the exception of malate plus glutamate; in this case, inhibition of O₂ uptake was more severe due to an effect of cyanide on aspartate aminotransferase. Azide and antimycin A gave similar inhibitions with all substrates. Subsequent addition of NAD had no effect with any substrate. Providing that oxalacetate accumulation was prevented, rotenone inhibited all NAD-linked substrates equally and caused ADP:O ratios to decrease by one-third. Addition of succinate to mitochondria oxidizing malate stimulated oxygen uptake, but adding citrate and α-ketoglutarate did not. These results indicate that there is no direct link between malic enzyme and the rotenone- and cyanide-resistant respiratory pathways, and that there is no need to postulate separate compartmentation of malic enzyme and the other NAD-linked enzymes in the matrix.     

Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism

The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin-resistant rate of O₂ uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone-resistant O₂ uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O₂ uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

Transport of calcium ions by Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.     

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.     

Developmental Changes in the NAD Content in Sugar-Beet Root Mitochondria and Their Effect on the Oxidative Activity of These Organelles

The NAD content was determined in mitochondria isolated from sugar-beet roots at various stages of plant development. A high NAD content (7.6 ± 0.9 nmol/mg mitochondrial protein) was observed in the mitochondria of actively growing roots of 80–95-day-old plants, but it decreased ca. twofold by the end of the first year of plant development, before the roots were harvested for storage. The mitochondria isolated from roots stored at low temperature for two to three months and those after five to eight days of regrowth during the second year of plant development manifested an even lower NAD content (2.2 ± 0.4 and 2.0 ± 0.5 nmol/mg protein, respectively). A drastic decrease in the NAD content in mitochondria from stored roots did not result from the impairment of the inner membrane of these organelles and was evidently regulatory in its nature. The effect of developmental changes in the intramitochondrial NAD content on the malate oxidation pattern was studied. In the mitochondria of stored roots, the low NAD content limited the rate of malate oxidation in state 3, because the addition to the reaction mixture of exogenous NAD, which can be transported to the mitochondrial matrix, promoted malate oxidation by 30–50%. Rotenone inhibited malate oxidation in the stored-root mitochondria by more than 70%; in this case, the rate of rotenone-resistant malate oxidation in these organelles increased by several times in the presence of exogenous NAD. In the mitochondria of the growing root, exo-genous NAD did not affect the rate of malate oxidation, and rotenone inhibited it only by 25–35%. The analysis of the data obtained here and the published evidence suggests the existence of a universal mechanism of respiration control and the regulation of the functional activity of plant mitochondria. This mechanism acts through a change in the NAD content in the organelle matrix. This NAD can be used in the course of plant development, e.g., during the transition of sugar-beet-root cells in the dormant state, when the respiration rate must decline.     

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.)

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.     

A malate-oxaloacetate shuttle linking cytosolic fatty acid α-oxidation to the mitochondrial electron transport chain in uncoupled fresh potato slices

The possible existence of a malonate-sensitive dicarboxylate-mediated electron shuttle between microsomal NAD-linked fatty acid α-oxidation and the mitochondrial electron transport chain in uncoupled fresh potato slices was investigated. Uncoupled slice respiration is inhibited by benzylmalonate and butylmalonate, inhibitors of dicarboxylate transport into mitochondria. Uncoupled slice respiration is also inhibited by rotenone, an indication of intramitochondrial NADH oxidation. Since fatty acid α-oxidation per se is rotenone insensitive, rotenone and benzylmalonate inhibition of the oxidation of carboxyl-labeled myristate in slices points to a dicarboxylic acid shuttle linking microsomal fatty acid a-oxidation with intramitochondrial NADH dehydrogenase.
Malonute inhibits both respiration and ¹⁴CO², release from carboxyl-labeled myristate in fresh uncoupled slices, as do inhibitors of dicarboxylate transport. Mitochondrial studies show that malonate inhibits malate oxidation but not malate dehydrogenase per se. Furthermore, malonate inhibits malate transport more severely than malate oxidation. Accordingly, mulonate inhibition of uncoupled slice respiration in the absence of tricarboxylic acid cycle activity is attributed to its interference with mitochondrial malate transport, and its consequent curtailment of a putative malate-OAA shuttle linked to cytosolic NAD-mediated fatty acid α-oxidation.     

Adenylate control of respiration in plants: the contribution of rotenone-insensitive electron transport to ADP-limited oxygen consumption by soybean mitochondria

The aim was to test the hypothesis that rotenone-insensive electron transport (bypass of complex I) may underlie rapid state 4 (ADP-limited) mitochondrial respiration. A comparison of mitochondria from soybean ( Glycine max L. cv. Bragg) cotyledons and nodules showed that ADP-sufficient (state 3) malate plus pyruvate oxidation by mitochondria from 7-day-old cotyledons was inhibited 50% by rotenone and state 4 rates were rapid, whereas nodule mitochondria were 80% inhibited by rotenone and had slower state 4 rates of malate plus pyruvate oxidation. Respiration of malate alone (pH 7.6) by cotyledon mitochondria was slow, especially in the absence of ADP; subsequent addition of pyruvate dramatically increased state 4 oxygen uptake concomitant with a rapid rise in mitochondrial NADH (determined by fluorimetry). Rotenone had no effect on this increased rate of state 4 respiration. The rate of malate oxidation by nodule mitochondria was relatively rapid compared with cotyledon mitochondria. The addition of pyruvate in state 4 caused a slow increase in matrix NADH and only a slight stimulation of oxygen uptake. Rotenone inhibited state 4 malate plus pyruvate oxidation by 50% in these mitochondria. From a large number of cotyledon and nodule mitochondrial preparations, a close correlation was found between the rate of state 4 oxygen uptake and rotenone-resistance. During cotyledon development increased rotenone-resistance was associated with an increase in the alternative oxidase. Addition of pyruvate to cotyledon mitochondria, during state 4 oxidation of malate in the presence of antimycin A, significantly stimulated O₂ uptake and also almost eliminated respiratory control. Such combined operation of the rotenone-insensitive bypass and the alternative oxidase in vivo will significantly affect the extent to which adenylates control the rate of electron transport.     

Influence of carboxylic acids on the stereospecific nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases of Leuconostoc mesenteroides

Leuconostoc mesenteroides increased its lactic acid production from glucose threefold when malic acid was added to the culture. This increase resulted also in a reduction of the ratio of d-lactic acid to l-lactic acid (31.5 to 1.23). Addition of malic acid increased 6.5-fold the specific activity of nicotinamide adenine dinucleotide (NAD)-linked l-lactate dehydrogenase and increased 3.2-fold that of NAD-linked d-lactate dehydrogenase. The Michaelis constant (K(m)) for NAD of the NAD-linked l-lactate dehydrogenase increased with the addition of malate, but no change was observed in the K(m) values for the respective d-enzyme. The effect of carboxylic acids on the NAD-linked l-lactate dehydrogenase activities was tested by using partially purified enzyme preparations from cells grown with glucose alone and from cells grown with glucose plus malate. Malate stimulated the l-enzyme and inhibited the d-lactate dehydrogenase. The NAD-linked l-lactate dehydrogenase exhibited the same activity bands on polyacrylamide gel electrophoresis whether the cell-free preparation originated from cells grown on glucose plus malate or on glucose as the sole carbon source. The NAD-linked d-lactate dehydrogenase, however, exhibited a different pattern of electrophoretic mobility, depending upon the source of origin of the cell-free preparation. The results suggest that malate has a stimulatory effect on the synthesis of both enzymes and may result in rearrangement of the protein structure of the d-lactate dehydrogenase. This rearrangement apparently makes the d-enzyme more susceptible to inhibition of catalytic activity. The l-lactate dehydrogenase, however, is stimulated not only in its synthesis but also in its activity. It is proposed that these effects are responsible for the regulation of lactic acid production.     

Evidence against tight channelling of NADH in hepatocytes

Tritiated substrates at tracer levels were incubated with rat hepatocytes plus 10 mM L-lactate, and the yields of tritium in glucose and water, as well as the tritium distribution on C-6 and C-4 of glucose, determined. Substrates of cytosolic type A NAD-linked dehydrogenases showed some preferential labeling of C-6 of glucose (the pathway involving type A malate dehydrogenase), whereas substrates of cytosolic type B NAD-linked dehydrogenases showed some preferential labeling of C-4 of glucose (the pathway involving type B glyceraldehyde-3P dehydrogenase). The results found are consistent with a classical diffusion model of NADH metabolism, and are at odds with the Srivastava hypothesis (based on isolated enzyme studies) which indicated that direct transfer of NADH can occur between many NAD-linked enzymes but only when they are of opposite (A or B) specificity.     

Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria.

Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.     

Calcium uptake by bovine epididymal spermatozoa is regulated by the redox state of the mitochondrial pyridine nucleotides

Immature caput epididymal sperm accumulate calcium from exogenous sources at a rate 2- to 4-fold greater than mature caudal sperm. Calcium accumulation by these cells, however, is maximal in the presence of lactate as external substrate. This stimulation of calcium uptake by optimum levels of lactate (0.8-1.0 mM) is about 5-fold in caput and 2-fold in caudal sperm compared to values observed with glucose as substrate. Calcium accumulation by intact sperm is almost entirely mitochondrial as evidenced by the inhibition of uptake by rotenone, antimycin, and ruthenium red. The differences in the ability of the various substrates in sustaining calcium uptake appeared to be related to their ability to generate NADH (nicotinamide adenine dinucleotide). Previous reports have documented that mitochondrial calcium accumulation in several somatic cells is regulated by the oxidation state of mitochondrial NADH. A similar situation obtains for bovine epididymal sperm since calcium uptake sustained by site III oxidation of ascorbate in the presence of tetramethyl phenylenediamine and rotenone was also stimulated by NADH-producing substrates, including lactate, and inhibited by substrates generating NAD+ (nicotinamide adenine dinucleotide, oxidized form). Further, calcium uptake by digitonin-permeabilized sperm in the presence of succinate was stimulated when NADH oxidation was inhibited by rotenone. The compounds alpha-keto butyric, valeric, and caproic acids, which generate NAD+, inhibited the maximal calcium uptake observed in the presence of succinate and rotenone, and the hydroxy acids lactate and beta-hydroxybutyrate reversed this inhibition. These results document the regulation of sperm calcium accumulation by the physiological substrate lactate, emphasize the importance of mitochondria in the accumulation of calcium by bovine epididymal sperm, and suggest that the mitochondrial location of the isozyme LDH-X in mammalian sperm may be involved in the regulation of calcium accumulation.     

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K_i values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K_i of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.     

The oxidation of tricarboxylic acid cycle intermediates, with particular reference to isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata leSueur.

The oxidation of exogenously added substrates has been studied in intact liver mitochondria isolated from the American eel, Anguilla rostrata. These data, coupled to determinations of the activity and localization of critical tricarboxylic acid (TCA) cycle enzymes, have been used to propose a pathway for the eel liver TCA cycle. (1) Isocitric, α-ketoglutaric, succinic, and malic acids are oxidized at essentially equivalent rates by eel mitochondria, with normal ADP:O and respiratory control ratios. No oxidation of citric, oxaloacetic, or pyruvic acids was detected when added alone or with malate, although oxaloacetic acid + pyruvic acid was oxidized but at a much reduced rate. (2) Radioactively labeled isocitrate was incorporated into at least α-ketoglutaric, succinic, and malic acids, indicating the eel liver TCA cycle is normal between isocitrate and malate. (3) No activity of the NAD-linked isocitrate dehydrogenase (IDH) could be detected, but NADP-IDH activities were higher in the mitochondria than cytosolic fractions. An active NADPH:NAD transhydrogenase was localized to the mitochondrial compartment. (4) These data suggest an important role for the NADP-IDH:transhydrogenase enzyme couple in eel liver TCA cycle function, and a pathway incorporating these ideas is proposed.     

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 α-ketoglutarate and malate as substrates. Whereas α-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.

     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.