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PubMed1987

Thermal inactivation of electron-transport functions and F0F1-ATPase activities.

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1987

The anesthetic nitrous oxide affects dioxygen utilization by bovine heart and bean seed mitochondrial particles.

Nitrous oxide affects dioxygen utilization by both bean seed and bovine heart submitochondrial particles when either succinate or reduced cytochrome c are used as substrates. Bovine heart particles exhibit reversible, dose-dependent partial inhibition of respiratory activity when exposed to N2O. Bean seed particle respiration is stimulated by low levels of N2O, but higher concentrations are inhibitory. These findings can be explained in terms of one locus of anesthetic action: cytochrome c oxidase, the terminal component of the mitochondrial respiratory chain. Alterations in respiration rates are expected to make important contributions to anesthesia in animals and to control of germination in plants.

Animals↗
PubMedDate not supplied

[Potential difference across subcellular particle membranes. II. Compensation method of measurement].

"Zero-loop" of the molecular potential transformer of submitochondrial particles (SMP) is separated from the remaining electron transfer chain by rotenone, and its e.m.f. ET=0,003+RT/2F in [NADP X H] [NAD+]/[NADP+] [NAD X H] volts is used in the compensative method of measurement of the potential difference across the SMP membrane (delta USMP). The phospholipid membrane, measuring the concentration of the penetrating anions in the solution contained SMP, is used as "zero-indicators". This concentration drops monotonically with increase in delta USMP. Delta USMP is equal to ET when the addition of substrates of transhydrogenase reaction with definite ET does not change the potential across phospholipid membrane.

Animals↗
PubMed1970

The surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure.

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.

Animals↗
PubMedDate not supplied

[Potential difference across the membrane of subcellular particles. IV. Study of the part of the respiratory chain from FAD to cytochrome c by the ion penetration technic].

Ability of mitochondria and submitochondrial particles (SMP) to generate a membrane potential on the addition of unpenetrating electron donors and acceptors and trimethylhydroquinone was used for specification of organization of the electron transfer chain. Ferri- and ferrocyanide added to mitochondria induce the membrane potential generation exchanging electrons only with cytochromes c or c1. Ferricyanide added to SMP induces the membrane potential taking electrons in three places: from NAD-H dehydrogenase, succinate dehydrogenase and in the presence of antimicyne A from cytochrome beta region. The difference of two sides of mitochondrial membrane is in agreement with the chemielectric hypothesis. According to this hypothesis protons from internal part of mitochondria are taken up to input H+-channel after electrons. That is why electrons come near the inner surface of the membrane and can be transferred from respiratory chain to water soluble acceptors. In output channels protons move under the action of intramembrane electrostatic field and in the direction of rising polarization of the medium. Protons are released to output channels while electrons are transferred to next carrier within the hydrophobic part of the membrane far from external surface of the membrane and cannot be transferred to water soluble acceptors.

Electron Transport↗
PubMed1994

Effect of aspartate and glutamate on the oxoglutarate carrier investigated in rat heart mitochondria and inverted submitochondrial vesicles.

Interaction of glutamate and aspartate with the oxoglutarate carrier was investigated in rat heart mitochondria or inverted submitochondrial particles. With mitochondria, glutamate and aspartate had no effect on the initial rate of oxoglutarate or malate uptake. With inverted submitochondrial vesicles, binding experiments indicated that aspartate bound to the oxoglutarate carrier on its matricial face and increased the affinity of the substrate binding site for malate but did not change the affinity for oxoglutarate. Glutamate had no effect on both substrate bindings. The dissociation constants of the binary substrate-carrier complexes on the matricial side were determined (1.28 +/- 0.15 mM for oxoglutarate and 2.22 +/- 0.26 mM for malate). These values, compared with those obtained previously on the cytosolic side of intact mitochondria, confirmed the asymmetry of the carrier in the native membrane (higher affinities on the cytosolic face). It is concluded that (1) aspartate and glutamate are not cytosolic effectors of the oxoglutarate carrier, (2) matricial aspartate is a positive effector of the binding of malate on the matricial side of the oxoglutarate carrier, and (3) such a characteristic may play a role in the regulation of the oxoglutarate carrier. Thus, it may be emphasized that (1) this observation is the first clear evidence of a well-defined 'sophisticated regulation' (allosteric) of a mitochondrial metabolite carrier, and (2) this regulation of the oxoglutarate carrier may have important consequences on the efficiency of reducing equivalent import in the matrix space by the malate-aspartate shuttle.

Animals↗
PubMed1988

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems.

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.

Animals↗
PubMed1998

Comparison of energization of complex I in membrane particles from Paracoccus denitrificans and bovine heart mitochondria.

The results of preliminary studies of the effects of energization on the catalytic and EPR properties of complex I in tightly coupled membrane vesicles of Paracoccus denitrificans (SPP) are presented. They are compared to those observed in submitochondrial particles from bovine heart (SMP). All signs of energization of complex I detected by EPR in SMP (uncoupler-sensitive splitting of the gz lines of the clusters 2 and a broadening of their gxy lines, a fast-relaxing, piericidin-sensitive ubiquinone-radical signal, and a broad signal around g = 1.94) were also observed with the bacterial enzyme. There were some prominent differences, though. The signal of the fast-relaxing radicals could be evoked both in the presence or absence of reduced clusters 2, suggesting that enhancement of its spin-relaxation rate is caused by coupling to another paramagnet. The signal was hardly affected by the presence of gramicidin. The slow-relaxing radical signal did not disappear upon anaerobiosis, but was detectable for at least another 30 s. The fast-relaxing signal vanished immediately upon anaerobiosis. The activity of the bacterial enzyme during oxidation of NADH by oxygen or reduction of NAD induced by succinate oxidation, was 5-6 times higher than that of the mitochondrial enzyme. Unlike the mitochondrial enzyme, the bacterial enzyme was not inactivated by incubation at 35 degrees C. The spin concentration of the NADH-reducible [2Fe-2S] cluster (1b) was half that of the clusters 2, indicating no difference with the mitochondrial enzyme.

Animals↗
PubMed1989

Evidence that coupling factor B is bound to the matrix side of the inner mitochondrial membrane.

Rotenone-sensitive NADH dehydrogenase activity and Lubrol stimulation of cytochrome oxidase activity were measured to assess the opposite membrane polarity of beef heart mitoplast and inside-out particle preparations. The ATP-Pi exchange activity of mitoplasts was not affected by their incubation at pH 8.9 in the presence of 5 mM EDTA (a treatment known to extract coupling factor B (F beta) from submitochondrial particles), nor was it stimulated by the addition of F beta to intact and alkaline treated mitoplast preparations. In contrast, the exchange activity of inside-out particles was decreased 18 fold by the alkaline/EDTA treatment and was almost completely restored by the addition of F beta to F beta-depleted particles. From these results it is concluded that in beef heart mitochondria, the coupling factor F beta is bound to the matrix-side of the inner mitochondrial membrane.

Adenosine Triphosphatases↗
PubMed1990

Demonstration of "naturally occurring mitochondrial antibodies" in family members of patients with primary biliary cirrhosis.

Sera from 81 healthy family members (including husbands) of 13 patients with primary biliary cirrhosis were tested by Western blotting against the antigen fractions M9 and M2 derived respectively from rat liver and beef heart mitochondria. Fifty-eight (70%) were positive and recognized four major (molecular weights 98 kD, 65 kD, 61 kD, 58 kD) and eight minor determinants (85 kD, 81 kD, 78 kD, 54 kD, 48 kD, 45 kD, 40 kD, 30 kD) labeled alpha-my. Each of the 58 sera recognized at least one of the four major polypeptides. In contrast, only 6% of 80 primary biliary cirrhosis patients had antibodies against one of these epitopes. Sera from 25 patients with different infectious disorders previously shown to react with submitochondrial particles by enzyme-linked immunosorbent assay also recognized at least one of the four major determinants. From these findings it was concluded that these antibodies may belong to the family of natural autoantibodies. Since they reacted with submitochondrial fractions, they were defined as "naturally occurring mitochondrial antibodies". The high incidence of "naturally occurring mitochondrial antibodies" in primary biliary cirrhosis-contact persons may be taken as indirect evidence for a contagious immunogenic agent circulating in the blood of primary biliary cirrhosis-patients. In contrast, the absence of "naturally occurring mitochondrial antibodies" in primary biliary cirrhosis-patients themselves implies that an underlying B-cell defect is responsible for this lack of antibody production. Considering the protective role of naturally occurring antibodies in general, this postulated B-cell defect could be a major factor in the etiopathogenesis of primary biliary cirrhosis.

Antibodies↗
PubMed1975

Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation.

Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex. It catalyzes ATP-Pi exchange and ATP hydrolysis. The exchange reaction is sensitive to uncouplers, rutamycin, valinomycin plus K-+, dicyclorexylcarboditmide, arsenate, azide, and adenylyl imidodiphosphate. It is also specific for ATP; ITP, GTP and UTP are essentially ineffective. Studies with the photoaffinity labeling uncoupler, 2-azido-4-nitrophenol (NPA), have shown that the mitochondrial uncoupler-binding sites are located exclusively in complex V. Complexes I, III and IV, which carry the three coupling sites of the respiratory chain, had negligible capacity for the binding of NPA, whereas the uncoupler-binding capacity of complex V appeared to be increased two- to threefold as compared to mitochondria. Complexes I, II, III, IV and V are obtained from the same batch of mitochondria by a simple fractionation procedure, which employs cholate, deoxycholate, ammonium acetate and ammonium sulfate. Studies with NPA have shown that mitochondria contain per milligram protein about 0.6 nmole of uniformly reacting uncoupler binding site. All of the uncouplers tested appeared to interact competitively with this site. Photoaffinity labeling with tritiated NPA has shown that a major portion of NPA binds to a polypeptide of molecular weight between 26,000 and 30,000. Other studies on the mechanism of uncoupling have shown that picrate is a membrane-impermeable uncoupler. It cannot uncouple mitochondria. However, it is an effective uncoupler of ATP synthesis and ATP-induced transhydrogenation or reverse electron transfer when used in conjunction with sonicated submitochondrial particles, which have an inside-out orientation of the inner membrane with respect to the medium. In these particles, picrate binds to the same uncoupler-binding site as NPA and other uncouplers. However, unlike the membrane-permeable uncouplers, picrate is a poor protonophore. It has a very small effect on the proton permeability of phosphorylating submitochondrial vesicles, even at two to three times the concentration needed for complete uncoupling. The increase in the proton permeability of submitochondrial vesicles caused by such high concentrations of picrate (500 mum) can be achieved with approximately 5 mum 2,4-dinitrophenol. At this concentration, dinitrophenol results in only about 20% uncoupling.

Adenosine Triphosphate↗
PubMed2006

Mitochondrial respiratory dysfunction and oxidative stress after chronic malathion exposure.

Malathion is a pesticide used on a large scale and with high potential risk for human exposure. However, it is reasonable to hypothesize that while the malathion is metabolizing reactive oxygen species (ROS) can be generated and subsequently there is onset of an oxidative stress in central nervous system (CNS) structures: hippocampus, cortex, striatum and cerebellum of intoxicated rats due to mitochondrial respiratory chain disfunctions. The present study was therefore undertaken to evaluate malathion-induced lipid peroxidation (LPO), superoxide production from sub-mitochondrial particles and the activity of complexes II and IV of the mitochondrial respiratory chain. Malathion was administered in doses of 25, 50, 100 and 150 mg malathion/kg. After malathion administration LPO increased in hippocampus and striatum. This was accompanied by an increase in the formation of superoxide in submitochondrial particles in the hippocampus. Complex IV suffered significant inhibition of its activity. We could demonstrate in this study that malathion induces oxidative stress and it could be due to inactivation of mitochondrial respiratory complexes.

Animals↗
PubMed1965

Observations on iron uptake, iron metabolism, cytochrome c content, cytochrome a content and cytochrome c-oxidase activity in regenerating rat liver.

1. Differential and density-gradient centrifugation were used to fractionate mitochondria and fluffy layer from normal and regenerating rat liver. The iron, cytochrome a and cytochrome c contents and cytochrome c-oxidase activity were studied as well as the uptake of (59)Fe into protein and cytochrome c. 2. A certain degree of heterogeneity was evident between the heavy-mitochondrial and light-mitochondrial fractions, and in their behaviour during liver regeneration. 3. The specific content of light-mitochondrial iron and cytochrome a was 1.3-1.4 times that of heavy mitochondria. Changes in cytochrome c-oxidase activity closely followed those of cytochrome a content during liver regeneration, but not for light mitochondria after 10 days. 4. Radioactive iron ((59)Fe) was most actively taken up by well-washed light mitochondria during early liver regeneration. After 22 days fluffy layer became preferentially labelled. This substantiates the view that fluffy layer partially represents broken-down mitochondria. 5. During early regeneration, light-mitochondrial fractions separated along a density gradient were about 3 times as radioactive, and showed distinct heterogeneity of (59)Fe-labelling, in contrast with near homogeneity for heavy mitochondria. 6. Immediately after partial hepatectomy fractions corresponding to density 1.155 were 5-10 times as radioactive as particles of greater density. The radioactivity decreased sharply after 6 days. 7. These particles of low density possessed higher NADH-cytochrome c-reductase (1.5-5-fold) and succinate-dehydrogenase (1.1-2-fold) activities than typical mitochondrial fractions. Their succinate-cytochrome c-reductase and cytochrome c-oxidase activities were slightly lower. 8. The results are discussed in relation to mitochondrial morphogenesis, and a possible route from submitochondrial particles is suggested.

Journal Article↗
PubMed1985

Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase.

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.

Animals↗
PubMed1976

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.

Adenosine Triphosphatases↗
PubMed1973

Regulation of Succinate Dehydrogenase in Higher Plants: II. Activation by Substrates, Reduced Coenzyme Q, Nucleotides, and Anions.

The effect of various agents on the activation of succinate dehydrogenase in cauliflower (Brassica oleracea) and mung bean (Phaseolus aureus) mitochondria and in sonicated particles has been investigated. Reduced coenzyme Q(10), inosine diphosphate, inosine triphosphate, acid pH, and anions activate the enzyme in mitochondria from higher plants in the same manner as in mammalian preparations. Significant differences have been detected in the behavior of plant and animal preparations in the effects of ATP, ADP, NADH, NAD-linked substrates, and of 2, 4-dinitrophenol on the state of activation of the dehydrogenase. In mammalian mitochondria ATP activates, whereas ADP does not, and the ATP effect is shown only in intact mitochondria. In mung bean and cauliflower mitochondria, both ATP and ADP activate and the effect is also shown in sonicated and frozen-thawed preparations. In sonicated mung bean mitochondria NADH causes complete activation, as in mammalian submitochondrial particles, but in sonicated cauliflower mitochondria activation by NADH is incomplete, as is also true of intact, anaerobic cauliflower mitochondria. Moreover, neither NAD-linked substrates nor a combination of these with NADH can fully activate the enzyme in cauliflower mitochondria. In contrast to mammalian mitochondria, succinate dehydrogenase is not deactivated in cauliflower or mung beam mitochondria under the oxidized conditions brought about by uncoupling of oxidative phosphorylation by 2,4-dinitrophenol.

Journal Article↗
PubMed1980

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase.

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.

Adenosine Triphosphatases↗
PubMed2002

Biochemical-clinical correlation in patients with different loads of the mitochondrial DNA T8993G mutation.

OBJECTIVE: To investigate the correlation between biochemical and clinical phenotype in 6 patients from 3 unrelated families with different mutation loads (heteroplasmy) of the T8993G mitochondrial DNA mutation associated with neuropathy, ataxia, and retinitis pigmentosa-Leigh syndrome. METHODS: We studied adenosine triphosphate (ATP) synthase activity (synthesis and hydrolysis) in platelet-derived submitochondrial particles and assessed mutant loads both in platelets used for biochemical analysis and in other available tissues. Biochemical and molecular results were correlated with clinical features. RESULTS: The rate of ATP hydrolysis was normal, but ATP synthesis was severely impaired (30% to 4% of residual activity) in patients harboring 34% to 90% mutant mitochondrial DNA, without any evidence of a threshold for the expression of this defect. There was little variation in heteroplasmy among tissues from each patient, but wider variability was detected in 2 mothers. Correlation of heteroplasmy and clinical and biochemical features suggested that ATP synthesis is defective at mutant loads as low as 34% and is extremely reduced at mutant loads above 80% when the phenotype is neuropathy, ataxia, and retinitis pigmentosa-Leigh syndrome. CONCLUSIONS: This study indicates a close relationship between tissue heteroplasmy, expression of the biochemical defect in platelets, and clinical involvement. The biochemical defect was greater than previously reported, and we found no evidence of a biochemical threshold. The uniform distribution of high mutant loads among our patients' tissues suggests a differential tissue-specific reliance on mitochondrial ATP synthesis.

Adenosine Triphosphate↗