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At least 37 records · Page 2Linked to original sources
PubMed1980

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS.

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by "aging" in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. "Aged" or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO(3), which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or "aged" submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO(3) (-), but inhibited by Cl(-), indicating that Cl(-) stimulation is a distinguishing property of the pea mitochondrial ATPase complex.

Journal Article↗
PubMed1980

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.

Adenosine Diphosphate↗
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↗
PubMed1979

Oxidative phosphorylation in pea cotyledon submitochondrial particles.

Mitochondria and submitochondrial particles (SMP) from pea cotyledons were shown to catalyze oxidative phosphorylation as measured by (32)Pi uptake into phosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomycin. Experiments with the adenine nucleotide translocator inhibitor atractyloside indicated the SMP were inside-out. Mersalyl completely inhibited ATP synthesis by SMP, and a separate experiment indicated that mersalyl has a direct effect on the ATPase complex. The kinetics of ATP synthesis indicated a high affinity for phosphate (K(m) = 0.18 millimolar). ADP kinetics gave a biphasic curve with K(m) values of about 4.8 and 160 micromolar. O(2) uptake and ATP synthesis had a pH maximum of 7.6 while the ratio of micromoles phosphate esterified to microatoms O(2) taken up was highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and O(2) uptake but stimulated the ATPase reaction. The SMP also catalyzed a slow ATP-phosphate exchange reaction.

Journal Article↗
PubMed1980

Effects of chronic ethanol consumption on the respiratory chain of rat liver submitochondrial particles.

Studies of hepatic submitochondrial particles, which provide an experimental system allowing direct measurements of electron transfer independent of substrate transport reactions, showed that chronic ethanol ingestion (36% calories, 40 days) lowered the specific respiratory rates associated with substrate oxidation. NADH oxidase activity was decreased about 40%, succinoxidase was decreased 25%, and oxidation in ascorbate mediated by phenazine methosulfate was decreased 20%. The content of dithionite-reducible cytochrome aa3 was decreased 38%, while that of cytochrome b was decreased 8%, and that of cytochromes c + c1 was decreased 14%. Steady state kinetic measurements indicated that the turnover number of cytochrome oxidase was unchanged, about 15 s-1 under uncoupled conditions with NADH as substrate. When electron flux to cytochrome c was maximal, cytochrome c was maintained in a more highly reduced state relative to cytochrome aa3 in submitochondrial particles from the ethanol-treated rat compared to those from the control rat. This finding is consistent with the greater decrease in cytochrome aa3 content relative to that of cytochrome c. The results indicate that the diminished content of cytochrome oxidase is one of the factors responsible for the lower respiration rates caused by chronic ethanol consumption.

Alcoholism↗
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↗
PubMed1996

Presence of an NAD(P)H dehydrogenase and A b-type cytochrome different from the respiratory chain in submitochondrial particles from human placenta.

Placental submitochondrial particles can synthesize progesterone. Both, progesterone synthesis and oxygen consumption were stimulated in the presence of succinate, NADH, or NADPH. NADPH reduced the cytochromes of the respiratory chain in a similar way as NADH. NADPH oxidation was not achieved by NADH-DH from the respiratory chain, and the presence of an NAD(P)H-DH is suggested. Submitochondrial particles contain at least two b-type cytochromes, one inhibited by antimycin when the electron donor is succinate, whereas the other is less sensitive to antimycin in the presence of pyridine nucleotides. The results suggest a connection between the respiratory chain and cytochrome P450, through an NAD(P)H-DH and a b-type cytochrome, both different from the respiratory chain.

Antimycin A↗
PubMed1984

Localization of a ferricyanide-reactive site of cytochrome b-c1 complex, possibly of cytochrome b or ubisemiquinone, at the outer face of submitochondrial particles.

When succinate oxidation by submitochondrial particles is blocked by antimycin, NoHOQnO or funiculosin, addition of ferricyanide restores oxygen uptake coupled to membrane potential generation. The effect of ferricyanide is abolished by mucidin or myxothiazol, as well as by KCN. The data strongly favor a cyclic redox loop mechanism in site 2 and show that either heme of the ferrous cytochrome b or ubisemiquinone formed in the QH2-oxidizing center of complex b-c1 is accessible to ferricyanide at the outer (M) side of the submitochondrial particle membrane.

Animals↗
PubMed1981

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

Adenosine Triphosphatases↗
PubMed1981

ADP-arsenate. Formation by submitochondrial particles under phosphorylating conditions.

Submitochondrial particles from beef heart mitochondria synthesize ADP-arsenate from ADP and arsenate when energized by succinate. The ADP-arsenate formed hydrolyzes rapidly and this is almost certainly the mechanism by which arsenate "uncouples" oxidative phosphorylation. When sufficient hexokinase is present, a substantial portion of the ADP-arsenate formed reacts with glucose to form glucose 6-arsenate and ADP. The glucose 6-arsenate thus formed hydrolyzes, at pH 7.5 and 30 degrees C, under the conditions used, with a rate constant of 5.5 X 10(-4) s-1 and is a substrate for glucose-6-phosphate dehydrogenase.

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↗
PubMed1993

The adenine nucleotide translocase modulates oligomycin-induced quenching of pyranine fluorescence in submitochondrial particles.

Incorporation of the fluorescent, nonpermeant pH indicator pyranine into submitochondrial particles (pyranine-SMP) permitted monitoring of intravesicular pH changes brought about by proton translocation due to oxidation of respiratory chain substrates or to hydrolysis of ATP. Addition of oligomycin to beef heart pyranine-SMP was followed by a pH-independent quenching of pyranine fluorescence. Quenching was influenced by the presence of adenine nucleotides both inside and outside the submitochondrial particles. The nature of the nucleotides required for quenching resembled the specificity of the adenine nucleotide translocase rather than F1-ATPase. Removal of F1 from pyranine-SMP by treatment of the particles with urea did not alter oligomycin-induced quenching. Atractyloside, a specific inhibitor of the adenine nucleotide translocase, prevented oligomycin-induced quenching when the inhibitor was coincorporated into submitochondrial particles with pyranine. Bongkrekic acid prevented or reversed the oligomycin-dependent quenching when added to pyranine-SMP either before or after oligomycin, respectively, but only when ATP was present within the particles. A mutant of Saccharomyces cerevisiae, lacking translocase genes, exhibited oligomycin-dependent fluorescence quenching which was not inhibited by bongkrekic acid. The results support the interpretation that oligomycin promotes sequestration of the fluorescent probe in a region of the submitochondrial particle, probably the F0F1 complex, that leads to a quenching of fluorescence. The observed quenching can be modulated in a way that suggests an interaction between the translocase and F0.

Adenosine Triphosphate↗
PubMed1999

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart.

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.

Animals↗
PubMed1970

Energy-linked Functions of Submitochondrial Particles Prepared from Mung Bean Mitochondria.

Submitochondrial particles from mung bean mitochondria (Phaseolus aureus) are able to catalyze an energy-linked reduced nicotinamide adenine dinucleotide-nicotinamide adenine dinucleotide phosphate transhydrogenase reaction supported by ATP or by aerobically generated high energy intermediates. The energy transfer pathway appears to differ from that utilized for oxidative phosphorylation.Mung bean submitochondrial particles will also reduce nicotinamide adenine dinucleotide by reversed electron transport from succinate or ascorbate tetramethyl-p-phenylenediamine. The energy requirement can be met by ATP or by aerobically generated high energy intermediates.A scheme for the energy transduction pathway in mung beans is postulated from the effects of inhibitors and uncouplers of energy transfer on transhydrogenase and reversed electron transfer reactions.

Journal Article↗
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↗
PubMed1999

-->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q1. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t1/2 < or = 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H+ decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting -->H+/2e- stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q1 reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same -->H+/2e- stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q1-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+.

Adenosine Diphosphate Ribose↗
PubMed1984

Comparison of cumene hydroperoxide- and NADPH/Fe3+/ADP-induced lipid peroxidation in heart and liver submitochondrial particles. Mechanisms of protection by succinate.

The NADPH/Fe3+ /ADP system stimulates lipid peroxidation both in rat liver and bovine heart submitochondrial particles, while cumene hydroperoxide is active only in rat liver submitochondrial particles. The lack of a peroxidizing effect of cumene hydroperoxide in heart submitochondrial particles was related to the absence of cytochrome P-450. When ubiquinones are extracted from rat liver and bovine heart submitochondrial particles, succinate can still partially protect the cumene hydroperoxide-induced lipid peroxidation but not the peroxidation induced by NADPH/Fe3+ /ADP. The protective effect of succinate in lipid peroxidation was referred either to the reduction of ubiquinones that can act as antioxidants in the NADPH/Fe3+ /ADP system, or to the reduction of cytochrome P-450 that acts as a peroxidase in the cumene hydroperoxide system.

Adenosine Diphosphate↗