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At least 19 recordsLinked to original sources
PubMed1985

Effect of iron deficiency on energy conservation in rat liver and skeletal muscle submitochondrial particles.

Submitochondrial particles prepared from liver and skeletal muscle of control and iron-deficient rats were examined for cytochrome content and for both energy-independent and energy-conserving functions. Liver submitochondrial particles appear quite resistant to iron deficiency with cytochrome content and electron-transferring or energy-conserving functions maintained at a level of 85% or better of normal. Iron-deficient skeletal muscle submitochondrial particles, in contrast, have decreased cytochrome content and only 15-20% of the normal capacity for oxidation through either complex I (NADH dehydrogenase) or complex II (succinate dehydrogenase). Energy-linked reactions which involve substrate oxidation/reduction (succinate----NAD+ reversed electron flow and succinate-driven energy-dependent transhydrogenation) are likewise markedly decreased, while ATP-driven energy-dependent transhydrogenation and mitochondrial ATPase are normal. Our data support the concept that iron deficiency leads to decreased electron-carrying capacity of iron-containing mitochondrial enzymes, with skeletal muscle being much more susceptible than liver, but that the mitochondria are otherwise normal with regard to energy conservation.

Adenine Nucleotides↗
PubMed1975

Synthesis of adenosine triphosphate by an artificially imposed electrochemical proton gradient in bovine heart submitochondrial particles.

Submitochondrial particles subjected to an artificially imposed electrochemical proton gradient consisting of a pH gradient (acid to base transition) and membrane potential (low to high K-+ transition in the presence of valinomycin) catalyzed the net synthesis of 2.5 nmol of [-32P]ATP per mg of protein from ADP and 32-Pi. Optimal reaction conditions included incubation of submitochondrial particles in malonate at pH 5.0 with valinomycin in the absence of added K-+, followed by a rapid transition to pH 7.5 and 100 mM K-+. ATP synthesis continued for about 6 s and was sensitive to uncouplers or oligomycin but insensitive to inhibitors of electron transport. Lower amounts of ATP were formed by either the pH gradient (25%) of K-+ gradient (15%) alone. These results demonstrate that an electrochemical gradient of protons can drive the synthesis of ATP by reversal of the proton-translocating ATPase independent of electron transport.

Adenosine Triphosphatases↗
PubMed1985

Current-voltage relationships for proton flow through the F0 sector of the ATP-synthase, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or leak pathways in submitochondrial particles.

Respiring submitochondrial particles from which the F1 sector of ATP-synthase was displaced generated a membrane potential in the range of 115-140 mV. Addition of oligomycin raised the membrane potential by approximately 40 mV. The lower membrane potential in particles with F1 displaced is attributed to partial dissipation of the proton electrochemical gradient as a consequence of proton flow through the open proton channels provided by the F0 sectors of the ATP-synthase. The characteristics of proton flow through the open F0 channels were studied by varying the rate of electron transport-driven proton translocation which permitted the establishment of a range of steady-state membrane potentials. Open F0 channels appeared to have a gated response to the membrane potential such that they were inoperative when the potential fell below approximately 110 mV. The membrane potential was measured as a function of respiratory rate in intact Mg-ATP submitochondrial particles that had been treated with low concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone. In general a linear dependence of membrane potential upon respiratory rate was observed except at the lowest concentrations of protonophore and highest respiratory rates, presumably because the effect of the protonophore was then offset by an increased rate of proton translocation driven by the respiratory chain. The effect of increasing concentrations of carbonylcyanide-p-trifluoromethoxyphenylhydrazone on the membrane potential of respiring submitochondrial particles was studied. It was found that equal amounts of the protonophore lowered the membrane potential to a lesser extent at lower values of the membrane potential. Treatment of Mg-ATP submitochondrial particles with oligomycin slightly increased (by approximately 10 mV) the size of the respiration-dependent membrane potential, but did not alter the profile of membrane potential as a function of succinate oxidation rate. The latter was controlled by titration with malonate. This result indicates that the F0 sector of the ATP-synthase does not significantly contribute to leak pathways in intact submitochondrial particles.

Adenosine Triphosphate↗
PubMed1986

Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles.

Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762-3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.

ATP Synthetase Complexes↗
PubMed1976

Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles.

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.

Adenosine Diphosphate↗
PubMed1978

Glucagon treatment stimulates the metabolism of hepatic submitochondrial particles.

Hepatic submitochondrial particles, prepared at neutral pH from rats pretreated with glucagon, exhibited stimulated rates of State 3 and uncoupled respiration when succinate or NADH were the substrates, but not when ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine were employed. Measurements of 8-anilino-1-naphthalenesulfonic acid fluorescence in the particles indicated that glucagon treatment resulted in a stimulation of energization supported by succinate respiration or ATP hydrolysis. Similarly, the energy-linked pyridine nucleotide transhydrogenase and reverse electron flow reactions driven by succinate oxidation or ATP were also stimulated. The results indicate that mitochondrial substrate transport is not the prime locus of glucagon action. It is suggested that the increased level of energization in particles prepared from glucagon-treated rats is a reflection of a stimulation of the respiratory chain, possibly between cytochromes b and c, and the ATP-forming reactions.

Adenosine Triphosphate↗
PubMed1976

Anion and amine uptake and uncoupling in submitochondrial particles.

1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KCl or NH4Cl. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-1-napthalene sulphonate cause a pH-independent, 50% uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 muM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two pHases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects; uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as Cl-, the uncoupling effect is independent of Cl-. Furthermore the amine uncoupling is markedly enhanced by lipophilic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.

Amines↗
PubMed1987

Prevention of lipid peroxidation by NAD(P)H in rat liver submitochondrial particles.

In rat liver submitochondrial particles both NADH and NADPH inhibit lipid peroxidation induced by cumene hydroperoxide. Concomitantly with the inhibition of lipid peroxidation, NADH and NADPH strongly stimulate the peroxidase activity of rat liver submitochondrial particles. Rotenone slightly prevents both the protective effect on malondialdehyde formation and peroxidase activity. The peroxidase activity of rat liver submitochondrial particles was attributed to the NAD(P)H-mediated reduction of mitochondrial cytochrome P-450 which can act upon hydroperoxides, by decomposing them to alcohols.

Alcohols↗
PubMed1979

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa.

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.

Adenosine Monophosphate↗
PubMed1987

Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin.

Yeast submitochondrial particles, in a Pi- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5-10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.

Adenosine Triphosphate↗
PubMed1985

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart.

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.

Adenosine Diphosphate↗
PubMed1977

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.

Cytochrome c Group↗
PubMed1976

A kinetic analysis of the changes in fluorescence on the interaction of 8-anilinonaphthalene-1-sulphonate with submitochondrial particles.

A comparison of the fluorescence change on the addition of 8-anilinonaphthalene-1-sulphonate to succinate-energized submitochondrial particles with that on the addition of succinate to submitochondrial particles incubated with 8-anilinonaphthalene-1-sulphonate shows that these changes in fluorescence may be explained solely in terms of 8-anilinonaphthalene-1-sulphonate binding. This comparison does not support the proposal of an 8-anilinonaphthalene-1-sulphonate-monitored change in the conformation of submitochondrial-particle membranes [Brocklehurst, Freedman, Hancock & Radda (1970) Biochem. J.116, 721-731]. The biphasic nature of the decrease in fluorescence, which was found to follow the addition of uncoupler to submitochondrial particles incubated with ATP or succinate, or of antimycin A to submitochondrial particles incubated with succinate, does not support the existence of 'aplectic' and 'symplectic' states of the mitochondrial membrane [Barrett-Bee & Radda (1972) Biochim, Biophys. Acta 267, 211-215].

Acetone↗
PubMed1979

Kinetics of the potential-sensitive extrinsic probe oxonol VI in beef heart submitochondrial particles.

The interaction of the potential-sensitive extrinsic probe oxonol VI with beef heart submitochondrial particles has been investigated under time resolved and equilibrium conditions. The time course of the probe absorption spectrum red shift induced by ATP or NADH injection into a suspension of submitochondrial particles in a dye solution is biphasic, consisting of a faster process described by a second-order rate law with k2 approximately 3 x 10(5) M-1 sec-1. For the ATP pulse experiments, the slower process follows first-order kinetics with k1 approximately 0.3 sec-1. In oxygen pulse experiments to an anaerobic dye-particle system, the slower process is not significantly developed due to rapid depletion of the oxygen, but the faster process follows second-order kinetics with the same rate of the oxygen, but the faster process follows second-order kinetics with the same rate constant as for the ATP and NADH cases. Evidence for permeation of the submitochondrial particle membrane by oxonol VI has been obtained; the slower process is interpretable as describing the permeation of the membrane bilayer. The results of the time-resolved work are consistent with a mechanism involving a redistribution of the dye from the bulk phase to the particle membrane. The value of the second-order rate constant for passive binding of the dye to submitochondrial particles is not compatible with a mechanism proposed to explain the microsecond probe response times in bilayer and excitable membrane experiments nor are such rapid signals observed in the oxonol VI-submitochondrial particle system.

Adenosine Triphosphate↗
PubMed1977

Protein distribution is submitochondrial particles after lipid peroxidation.

Three fractions of submitochondrial particles were isolated by differential centrifugation of mitochondrial inner membranes incubated with 1 mM ascorbate. Proteins were separated by polyacrylamide gel electrophoresis and their pattern was found to be different for the three fractions. These results are in agreement with previous work which showed the dissimilar enzyme distribution and chemical composition of these submitochondrial particles and pointed out the heterogeneity of the inner mitochondrial membrane.

Animals↗