Effect of Dequalinium on Respiration and the Inner Membrane Permeability of Rat Liver Mitochondria

The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V₃) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H₂O₂ production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed.     

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles

A kinetic study on ubiquinol-cytochrome f reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c₁ complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a K_m for ubiquinol-1 of 13.4 μM in mitochondria and of 24.6 μM in the isolated cytochrome b-c₁ complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent K_m values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the V_max and the K_m for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.     

Mitochondria-targeted Cytochrome P450 2E1 Induces Oxidative Damage and Augments Alcohol-mediated Oxidative Stress

The ethanol-inducible cytochrome P450 2E1 (CYP2E1) is also induced under different pathological and physiological conditions. Studies including ours have shown that CYP2E1 is bimodally targeted to both the endoplasmic reticulum (microsomes) (mc CYP2E1) and mitochondria (mt CYP2E1). In this study we investigated the role of mtCYP2E1 in ethanol-mediated oxidative stress in stable cell lines expressing predominantly mt CYP2E1 or mc CYP2E1. The ER+ mutation (A2L, A9L), which increases the affinity of the nascent protein for binding to the signal recognition particle, preferentially targets CYP2E1 to the endoplasmic reticulum. The Mt+ (L17G) and Mt++ (I8R, L11R, L17R) mutant proteins, showing progressively lower affinity for signal recognition particle binding, were targeted to mitochondria at correspondingly higher levels. The rate of GSH depletion, used as a measure of oxidative stress, was higher in cells expressing Mt++ and Mt+ proteins as compared with cells expressing ER+ protein. In addition, the cellular level of F₂-isoprostanes, a direct indicator of oxidative stress, was increased markedly in Mt++ cells after ethanol treatment. Notably, expression of Mt++ CYP2E1 protein in yeast cells caused more severe mitochondrial DNA damage and respiratory deficiency than the wild type or ER+ proteins as tested by the inability of cells to grow on glycerol or ethanol. Additionally, liver mitochondria from ethanol-fed rats containing high mt CYP2E1 showed higher levels of F₂-isoprostane production. These results strongly suggest that mt CYP2E1 induces oxidative stress and augments alcohol-mediated cell/tissue injury.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

The role of the ATP-sensitive potassium channel in the activation of the K+ cycle in rat liver mitochondria

It is known that the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) plays a key role in protecting myocardium during ischemia. We have suggested that the mechanism of this protection is associated with the potassium cycle in mitochondria. In this paper, for the first time, a direct proof was obtained of the existence of a cycle of potassium ions in rat liver mitochondria that are associated with the functioning of mitoK_ATP. Activation of the cycle was recorded by optical density changes of mitochondrial suspension in the form of two or three swelling-contraction waves of the organelles. Using activators and inhibitors of mitoK_ATP we showed that a significant role in the potassium cycle belongs to the channel. It was found that in vitro sildenafil has a direct effect on mitoK_ATP, being its activator. The results obtained indicate that the cardioprotective effect of sildenafil observed previously is associated with the activation of mitoK_ATP. In order to study the structure and volume changes of mitochondria in various stages of the cycle in the presence of potassium channel modulators, the electron microscopy studies of mitochondria preparations were carried out. A correlation between the optical density decrease of mitochondrial suspension and the swelling of mitochondria was revealed. The data obtained in this study suggest participation of mitoK_ATP in the protection of tissues from hypoxic damage.     

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Köhler P. B.,Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of ¹⁴CO₂ from 1,4-¹⁴C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg²⁺ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap₅A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO₂ and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO₂ or propionate. This system is inhibited in flukes incubated in vitro with mebendazole.It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.     

Attenuation of sugar cataract by ethyl pyruvate

The effects of fluoxetine on the oxidative phosphorylation of mitochondria isolated from rat brain and on the kinetic properties of submitochondrial particle F₁F₀-ATPase were evaluated. The state 3 respiration rate supported by pyruvate + malate, succinate, or ascorbate + tetramethyl-p-phenylenediamine (TMPD) was substantially decreased by fluoxetine. The IC₅₀ for pyruvate + malate oxidation was 0.15 mM and the pattern of inhibition was the typical one of the electron-transport inhibitors, in that the drug inhibited both ADP- and carbonyl cyanide m-chlorophenylhydrazone (CCCP)-stimulated respirations and the former inhibition was not released by the uncoupler. Fluoxetine also decreased the activity of submitochondrial particle F₁F₀-ATPase (IC₅₀ 0.08 mM) even though K_0.5 and activity of Triton X-100 solubilized enzyme were not changed substantially. As a consequence of these effects, fluoxetine decreased the rate of ATP synthesis and depressed the phosphorylation potential of mitochondria. Incubation of mitochondria or submitochondrial particles with fluoxetine under the conditions of respiration or F₁F₀-ATPase assays, respectively, caused a dose-dependent enhancement of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. These results show that fluoxetine indirectly and nonspecifically affects electron transport and F₁F₀)-ATPase activity inhibiting oxidative phosphorylation in isolated rat brain mitochondria. They suggest, in addition, that these effects are mediated by the drug interference with the physical state of lipid bilayer of inner mitochondrial membrane.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.     

Dissecting the molecular mechanism by which NH2htau and Aβ1-42 peptides impair mitochondrial ANT-1 in Alzheimer disease

To find out whether and how the adenine nucleotide translocator-1 (ANT-1) inhibition due to NH₂htau and Aβ1-42 is due to an interplay between these two Alzheimer's peptides, ROS and ANT-1 thiols, use was made of mersalyl, a reversible alkylating agent of thiol groups that are oriented toward the external hydrophilic phase, to selectively block and protect, in a reversible manner, the –SH groups of ANT-1. The rate of ATP appearance outside mitochondria was measured as the increase in NADPH absorbance which occurs, following external addition of ADP, when ATP is produced by oxidative phosphorylation and exported from mitochondria in the presence of glucose, hexokinase and glucose-6-phosphate dehydrogenase. We found that the mitochondrial superoxide anions, whose production is induced at the level of Complex I by externally added Aβ1-42 and whose release from mitochondria is significantly reduced by the addition of the VDAC inhibitor DIDS, modify the thiol group/s present at the active site of mitochondrial ANT-1, impair ANT-1 in a mersalyl-prevented manner and abrogate the toxic effect of NH₂htau on ANT-1 when Aβ1-42 is already present. A molecular mechanism is proposed in which the pathological Aβ-NH₂htau interplay on ANT-1 in Alzheimer's neurons involves the thiol redox state of ANT-1 and the Aβ1-42-induced ROS increase. This result represents an important innovation because it suggests the possibility of using various strategies to protect cells at the mitochondrial level, by stabilizing or restoring mitochondrial function or by interfering with the energy metabolism providing a promising tool for treating or preventing AD.     

DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement     

THE ACCUMULATION AND THE RELEASE OF DIVALENT CATIONS ACROSS MITOCHONDRIAL MEMBRANES

Accumulated divalent cations and phosphate (P₁) in isolated bean mitochondria are released by conditions which inhibit respiration, including anaerobiosis and KCN, or by conditions which divert conserved energy from divalent cation uptake. These include ATP synthesis, K^T transport in the presence of valinomycin, and the presence of the uncouplers, 2,4-dinitrophenol and oleic acid. The results indicate that plant mitochondria are not permanent deposit sites for divalent cation and P₁ salts but, rather, function as temporary sequestering sites for these ions. It is suggested that mitochondria may play a role in the control of the movement as well as a regulation of the concentrations of these ions within the cell.     

In vitro incorporation of ( 3 H) methyl choline into phosphatidyl choline of rat liver subcellular fractions

Incubation of a rat liver total homogenate with radioactive choline and subsequent isolation of subcellular fractions, at different times, showed similar patterns of labeling. Incubation of microsomes, mitochondria and purified nuclei isolated from rat liver, showed that all fractions were able to incorporate the precursor into phosphatidyl choline. The specific activity was higher in mitochondria and increased in all cases with added supernatant. The addition of microsomes to mitochondria diminished the incorporation of label. Contamination of mitochondria by microsomes, was negligible as shown by undetectable amounts of cytochrome P₄₅₀, while NADPH₂ cytochrome c reductase showed a 10% contamination. A certain amount of radioactivity was incorporated in the absence of ATP and oxidizable substrates due to the presence of substrates and cofactors in the fraction and/or the supernatant. Labeled fractions reincubated with unlabeled choline, showed no loss of radioactivity, proving that incorporation was not due to simple exchange processes. It is concluded that although rat liver mitochondria can acquire part of their own provision of phosphatidyl choline by transference from microsomes, all organelles and specially mitochondria, can independently synthesize this phospholipid.     

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.)

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H₂O₂ (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α‐glycerophosphate, malate, and a combination of malate with another NAD‐linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca²⁺‐ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Structural and functional features of mitochondria in statocytes of soybean root under microgravity conditions

A study of the influence of microgravity and ethylene on the morphology and ultrastructural organization of mitochondria in root apex statocytes of 6-day old soybean seedlings that traveled on the Columbia space station on the STS-87 mission is presented. The seedlings were grown in hermetically sealed canisters in the presence of KMnO₄ to neutralize ethylene. It is established that, independent of KMnO₄ treatment, most of the mitochondria in the cells of the spaceflight seedlings were characterized by a round or oval shape and electronically transparent matrix, whereas in the seedlings in the ground controls the organelles were distinguished by a characteristic polymorphism, large dimensions, and higher electronic density of the matrix. One possible mechanism that could be responsible for the morphological and ultrastructural arrangement of the organelles in the process of adaptation of the seedlings to conditions of microgravity is discussed.     

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp_ha and Fp_hf and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp_hf is reduced more slowly than Fp_ha by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp_m. The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp_1f. In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.