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PubMedDate not supplied

Subcellular localization and properties of adenosine diphosphatase in human placenta.

It was found that mitochondria from human placenta exhibited an ADPase activity with the following characteristics. The enzyme responsible for this activity was associated with the inner mitochondrial membrane. It was not released by treatment of the submitochondrial particles with solutions of high ionic strength. Maximal ADP hydrolysis was reached at pH 8. Specific inhibitors for alkaline phosphatase (L-phenylalanine), myokinase (P1,P5-di(adenosine-5')pentaphosphate), or 5'-nucleotidase (concanavalin A) did not decrease ADP hydrolysis. ATP synthesis from ADP by myokinase was about 13 nmol/mg/min, whereas ADP hydrolysis reached values around 500 to 550 nmol/mg/min, indicating that a myokinase-H+ATPase combination could not account for the observed rates of ADP hydrolysis. The activity was stimulated by Mg2+, but high concentrations of this cation produced inhibition. High ADP concentrations did not inhibit ADPase activity. Kinetic measurements of the activity in the submitochondrial particles showed that the true substrate was ADP-Mg. The kinetic studies showed V(app) values of 476 and 270 nmol/mg/min, and Kmapp values of 416 and 8.7 microM.

Adenine Nucleotides↗
PubMed1997

Isolation and comparative studies of mitochondrial F1-ATPase from rat testis and beef heart.

The isolation and properties of F1-mitochondrial ATPase from rat testis are described. The isolation medium involves a chloroform extraction, and it is suitable even with small amounts of starting material that have a relatively low specific activity as in the case of rat testis submitochondrial particles. The isolated enzyme from rat testis had a specific activity of 30-45 mumol Pi/min/mg protein, which could be increased up to 90 mumol Pi/min/mg protein only in the presence of bicarbonate and maleate. The isolated enzyme represented less than 0.6% of the initial membrane proteins. It exhibited a typical five-band pattern in sodium dodecyl sulfate gel electrophoresis. However, it showed a ratio of subunits alpha:beta higher than the heart enzyme; its significance is unknown. The purified enzyme was cold labile and inhibited by natural ATPase inhibitor protein from bovine heart mitochondria and by dicyclohexylcarbodiimide. The results presented suggest that the low ATPase activity of testis submitochondrial particles is due to a reduced content of the F1-ATPase.

Adenosine Triphosphate↗
PubMed1987

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria.

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.

Animals↗
PubMed1988

Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase.

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Primary biliary cirrhosis: further biochemical and immunological characterization of mitochondrial antigens.

The nature of mitochondrial PBC-related antigens has been investigated with radioimmunoassay (RIA) and immunoblotting methods. The major antigen(s) was located by RIA in beef heart mitochondria, submitochondrial particles, chloroform-extracted F1-ATPase and Complex III. Cross-competition RIA experiments showed that the same antigen is present in all the above samples but at different concentrations. The antigen is not present in purified F1-ATPase, cytochrome oxidase, the oligomycin sensitivity conferring protein (OSCP), Factor6, or the Transhydrogenase. Immunoblot analysis of the above mitochondrial proteins revealed two PBC-related antigens (apparent molecular weights of 70 KD and 60 KD) whose distribution in the various proteins and protein complexes correlated well with the antigens determined by RIA. Immunoblot analysis of mitochondrial antigens was carried out using sera from normal subjects and from patients with PBC and with different autoimmune diseases (AID). Only PBC sera reacted with the 70 KD and 60 KD antigens. The PBC antigen detected by RIA in submitochondrial particles and the chloroform-F1-ATPase could be blocked by Mersalyl, suggesting its relationship to the mitochondrial 'M2' antigen. Furthermore, the antigenicity of the 70 KD peptide was shown by immunoblotting to be dependent upon mercaptoethanol. Thus, not only is the antigenicity of the 70 KD component dependent on a sulphur group, but the sulphur must be in the reduced form.

Adult↗
PubMed1982

Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol.

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In soluble F1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP in ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.

Adenosine Diphosphate↗
PubMed1975

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.

Animals↗
PubMed2002

Inhibition of glutamate release via recovery of ATP levels accounts for a neuroprotective effect of aspirin in rat cortical neurons exposed to oxygen-glucose deprivation.

BACKGROUND AND PURPOSE: Aspirin is preventive against stroke not only because of its antithrombotic properties but also by other direct effects. The aim of this study was to elucidate its direct neuroprotective effects. METHODS: Viability parameters, glutamate release and uptake, and ATP levels were measured in cultured cortical neurons exposed to oxygen-glucose deprivation (OGD). In addition, ATP levels and oxygen consumption were studied in isolated brain mitochondria or submitochondrial particles. RESULTS: Aspirin inhibited OGD-induced neuronal damage at concentrations lower (0.3 mmol/L) than those reported to act via inhibition of the transcription factor nuclear factor-kappaB (which are >1 mmol/L), an effect that correlated with the inhibition caused by aspirin on glutamate release. This effect was shared by sodium salicylate but not by indomethacin, thus excluding the involvement of cyclooxygenase. A pharmacological dissection of the components involved indicated that aspirin selectively inhibits the increase in extracellular glutamate concentration that results from reversal of the glutamate transporter, a component of release that is due to ATP depletion. Moreover, aspirin-afforded neuroprotection occurred in parallel with a lesser decrease in ATP levels after OGD. Aspirin elevated ATP levels not only in intact cortical neurons but also in isolated brain mitochondria, an effect concomitant with an increase in NADH-dependent respiration by brain submitochondrial particles. CONCLUSIONS: Taken together, our present findings show a novel mechanism for the neuroprotective effects of aspirin, which takes place at concentrations in the antithrombotic-analgesic range, useful in the management of patients with high risk of ischemic events.

Adenosine Triphosphate↗
PubMed1976

[Kinetic and structural characteristics of succinate dehydrogenase components reacting with natural and artificial electron acceptors].

A new catalitic activity of soluble succinate dehydrogenase, i.e. the reduction of low (20-200 muM) concentration of ferricyanide in the presence of succinate is described. The apparent Km value for the acceptor is about 200 muM. The turnover numbers of the enzyme measured in this reaction, with PMS as an electron acceptor and in the system reconstituted from soluble enzyme and alkali-treated submitochondrial particles (succinate oxidase) are found to be almost the same. The new succinate. ferricyanide reductase activity is very sensitive to oxygen, high (3 mM) ferricyanide concentration and mercaptide-forming agents. When the enzyme is stored under aerobic conditions the loss of this activity occurs according to the first-order kinetics with the same rate constants as the reconstitutive activity decreases. The rate constants both for ferricyanide reductase and reconstitution decay do not depend on pH within the range of 6,5--7,5 (k = 8.10(-2) min-1) and increase dramatically at pH 8,5 (K = 4.10(-1) MIN-1). When these two activities are lost after oxygen exposure the PMS-reductase fall down to about 50% of its original activity. The new ferricyanide reductase is found only in the soluble preparation of the enzyme succinate: cytochrome c reductase, succinate dehydrogenase of submitochondrial particles and reconstituted succinate oxidase do not interact with low concentrations of ferricyanide. The treatment of the enzyme after inactivation by oxygen exposure with sulfide ion--iron--mercaptoethanol mixture followed by Sephadex filtration completely restores the original reconstitutive, ferricyanide and PMS reductase activities. The hypothesis is suggested that succinate dehydrogenase contains at least two red-ox centers reacting with electron acceptors. The first one is located in hydrophylic environment (mitochondrial matrix) being accessible for high concentrations of ferricyanide. The second one (iron--sulfur complex, Hipip-type) is responsible for ferricyanide reductase activity described, being located intramembraneously and involved in the electron transfer between dehydrogenase and the rest of the respiratory chain.

Animals↗
PubMed1988

Heterogeneity of antimitochondrial antibodies with the M2-M4 pattern by immunofluorescence as assessed by Western immunoblotting and enzyme linked immunosorbent assay.

Seventy seven sera with antimitochondrial antibody exhibiting the M2-M4 pattern in immunofluorescence (56 from primary biliary cirrhosis (PBC), 21 from non-primary biliary cirrhosis patients) were studied by the combined use of Western immunoblotting with beef heart mitochondria and an enzyme linked immunosorbent assay (ELISA) with beef heart submitochondrial particles. Forty seven sera (10 without autoantibodies and 37 with different auto-antibodies) were included as controls. By immunoblotting, seven mitochondrial peptides reacting with antimitochondrial antibody positive sera were detected. These were of molecular weight 74 kD, 58 kD, 55 kD, 52 kD, 51 kD, 46 kD, and 43 kD. All primary biliary cirrhosis sera and 71% of antimitochondrial antibody-positive non-primary biliary cirrhosis sera reacted with one or more of these peptides, while none of the 47 antimitochondrial antibody negative sera reacted in immunoblotting. The 74 kD band was the most frequently detected (84% of primary biliary cirrhosis and 57% of non-primary biliary cirrhosis cases). All the primary biliary cirrhosis sera which failed to react with this peptide, showed a positive reaction with that of molecular weight 52 kD. 67/77 (87%) immunofluorescence antimitochondrial antibody positive sera reacted in the ELISA test (93% of primary biliary cirrhosis and 71% of non-primary biliary cirrhosis cases). All the 47 immunofluorescence antimitochondrial antibody negative sera were confirmed negative by ELISA. The ELISA values correlated with the immunofluorescence titres (p less than 0.05). By comparison of the results obtained by these two techniques, it emerged that the ELISA test (using our preparation of submitochondrial particles) was not able to detect the antibody directed against the mitochondrial peptide of 52 kD, which thus seems to be different from the other specificities.

Animals↗
PubMed1984

Rotational diffusion of the ADP/ATP translocator in the inner membrane of mitochondria and in proteoliposomes.

The ADP/ATP translocator was selectively labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide in beef heart mitochondria, as reported previously for submitochondrial particles (Müller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S. (1982) J. Biol. Chem. 257, 1117-1120). The EMA binding was completely inhibited by carboxyatractylate. 0.7-1.1 molecules of EMA conjugated with 1 molecule of the dimeric translocator with Mr approximately 65,000. The EMA binding decreased [14C]ADP uptake by about approximately 25%. The EMA-labeled translocator bongkrekate complex was purified and reconstituted in liposomes by removing Triton X-100 with Amberlite XAD-2. The liposomes were composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin and the lipid to protein ratio by weight was (L/P) = 60. Rotational diffusion of the ADP/ATP translocator around the membrane normal was measured in reconstituted proteoliposomes and in the mitochondrial inner membranes by observing the flash-induced absorption anisotropy, r(t), of EMA. In proteoliposomes with L/P = 60, the translocator was rotating with an approximate average rotational relaxation time of phi congruent to 246 microseconds and a normalized time-independent anisotrophy [r3/rr(0)]min congruent to 0.55. In intact mitochondria, values of phi congruent to 405 microseconds and r3/rr(0) congruent to 0.79 were obtained. The higher value of r3/rr(0) in mitochondria compared with proteoliposomes indicates the co-existence of rotating and immobile translocator (phi greater than 20 ms) in the inner mitochondrial membrane. Based on the assumption that all the translocator is rotating in the lipid-rich proteoliposomes, the population of the mobile translocator at 20 degrees C was calculated to be approximately 47%. By removing the outer membrane, the mobile population was increased to approximately 70% in mitoplasts, while approximately 53% of the translocator was rotating in submitochondrial particles. The above results indicate a significant difference in protein-protein interactions of the ADP/ATP translocator in the different types of inner membranes of mitochondria. The immobile population of the translocator could be due to nonspecific protein aggregates caused by the very high concentration of proteins in the inner membrane of mitochondria (L/P approximately 0.4).

Adenosine Diphosphate↗
PubMed1977

Cold lability of membrane-bound F1-ATPase.

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.

Adenosine Triphosphatases↗
PubMed1980

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy.

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.

Animals↗
PubMed1987

Activity of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation.

Cytochrome-c oxidase proteoliposomes containing internally trapped cytochrome c can turn over on internal or external cytochrome c. At low TMPD levels the internal activity is significantly lower than the external activity as the functional internal cytochrome c is not fully reduced in the steady state. Increasing TMPD concentration increases the internal rate to equal that of the external enzyme. Internal activity results in the accumulation of TMPD+. Valinomycin increases this accumulation and subsequently FCCP decreases it. In the presence of excess external cytochrome c, the effects of these ionophores are reversed. The internally-facing enzyme is thus capable of generating a delta mu H+ in proteoliposomes as well as in submitochondrial particles.

Animals↗
PubMed1987

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex.

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.

Anaerobiosis↗
PubMed1982

[Effects of lipids on the activity of soluble mitochondrial pyrophosphatase and its conversion to the membrane-bound form].

The effects of lipids on the activity of soluble and membrane-bound pyrophosphatase from beef heart mitochondria were studied. An addition of total mitochondrial lipid, phosphatidyl choline, phosphatidyl ethanolamine or cardiolipin resulted in stimulation of the enzymatic activity and an increase in thermal stability of the soluble enzyme. The maximal activating effect was exerted by the total mitochondrial lipid and phosphatidyl choline. The electrophoretic data suggest that phosphatidyl choline is a component of membrane pyrophosphatase. Preincubation of the soluble enzyme with phosphatidyl choline converted the enzyme into a membrane form, which is capable to carry out the energy-dependent synthesis of PPi in submitochondrial particles.

Animals↗
PubMed1993

Submitochondrial localization of the NAD+ glycohydrolase. Implications for the role of pyridine nucleotide hydrolysis in mitochondrial calcium fluxes.

The submitochondrial location of the NAD+ glycohydrolase (NADase) and its role in mitochondrial ADP-ribosyl transfer reactions were investigated in isolated rat liver mitochondria. The NADase catalyzes the hydrolysis of NAD+ to ADP-ribose and nicotinamide. Hydrolysis of intramitochondrial NAD+ has been suggested to be the first step in a nonenzymatic mono(ADP-ribosylation) by free ADP-ribose of an inner membrane-associated acceptor peptide and that this protein is involved in regulating calcium efflux from mitochondria during exposure to oxidants. The results of the present study indicate that mitochondrial NADase activity lies outside the matrix space, however. This was determined by assessing the rates of hydrolysis of externally added NAD+ by intact mitochondria and comparing these rates with those obtained when the mitochondria were permeabilized with detergent. No significant difference was observed in the rate of NAD+ hydrolysis when detergent was added indicating that NAD+ hydrolysis by mitochondria does not necessitate its access to the matrix space. The submitochondrial location of the NADase was investigated further by digitonin titration of isolated mitochondria. Digitonin titration of the mitochondria released NADase activity with the outer membrane marker, monoamine oxidase. The digitonin titration data also suggest that the outer membrane is the exclusive location of the NADase. The incorporation of radioactive label derived from [3H]NAD+ into submitochondrial particles proceeds at comparable rates in the absence or presence of NADase activity, indicating that the production of free ADP-ribose is not necessary for intramitochondrial ADP-ribosyl transfer reactions. Thus the conclusions of this study suggest that due to the submitochondrial location of the NADase, it does not participate in intramitochondrial pyridine nucleotide hydrolysis or nonenzymatic mono(ADP-ribosylation) during oxidative stress in mitochondria.

Adenosine Diphosphate Ribose↗