Electron Transport Particles Research Articles

Calcium-binding ATPase inhibitor protein of bovine heart mitochondria. Role in ATP synthesis and effect of Ca2+.

Submitochondrial particles (A particles) and phosphorylating electron-transport particles (ETPH) were prepared from bovine heart mitochondria. The A particles either were supplemented with or were depleted of the mitochondrial calcium-binding ATPase inhibitor protein (CaBI). The CaBI-depleted A particles still retained the Pullman-Monroy ATPase inhibitor protein (PMI), and the other particles all contained both CaBI and PMI. ATP synthase and ATPase activities of the particles were measured in similar reaction mixtures by luminescence of firefly luciferin-luciferase. Succinate was the respiratory substrate, and the adenylate kinase inhibitor P1, P5-di(adenosine-5') pentaphosphate was obligatory. The ATP synthase activity of CaBI-depleted A particles was 30-40% of that of the A and ETPH particles, and its ATPase activity was 7-8 times greater. Reconstitution of the CaBI-depleted A particles with CaBI restored the original ATP synthase and ATPase activities. ATP synthase activity rose about 1.7-fold when A particles were supplemented with additional CaBI and ATPase activity dropped to 9% of the original. Varying Ca2+ levels had little or no effect on the ATP synthase and ATPase activities of the CaBI-depleted A particles. In contrast, ATP synthase activity of the other particles was decreased by as much as 70% at the optimal Ca2+ concentration of 1 microM, and the ATPase activity of the A and EPTH particles rose concomitantly by 7-8-fold. The ATP synthase and ATPase activities of all the particles in microM Ca2+ became like those of the CaBI-depleted A particles. These changes were reversible; normal activities were restored as Ca2+ concentrations were raised above 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetraphenylborate potentiates the respiratory inhibition by the dopaminergic neurotoxin MPP + in both electron transport particles and intact mitochondria

The cytotoxicity of 1-methyl-4-phenylpyridinium (MPP +) is believed to arise as a consequence of its time- and energy-dependent accumulation inside mitochondria, followed by inhibition of electron transport at Complex I of the respiratory chain. Consistent with our proposal that the accumulation of MPP + represents a passive Nernstian transport into mitochondria in response to the transmebrane electrochemical potential gradient, tetraphenylborate (TPB −) was found to accelerate the onset of the respiratory inhibition by MPP + on intact mitochondria. Moreover, the ultimate level of inhibition reached was unexpectedly also increased. The latter is now explained by our finding that TPB − elicits a 12-fold enhancement of MPP + inhibition of respiration in electron transport particles. It is suggested that TPB − facilitates access of MPP + to its intramembrane site of inhibitory action in Complex I.

Inhibition of Mitochondrial F1-ATPase Activity by an Anti-α Subunit Monoclonal Antibody Which Modifies Interactions Between Catalytic and Regulatory Sites

A monoclonal antibody, 7B3, specific to the alpha subunit of the mitochondrial ATPase-ATP synthase inhibited the rate of ATP hydrolysis by either soluble F1 or electron transport particles up to a maximum of 75%. However, 7B3 did not modify the rate of ITP hydrolysis. In addition, the apparent Km for MgATP extrapolated at high ATP concentrations had the same value in the absence as in the presence of 7B3. The antibody did not change the inactivation rate of F1-ATPase induced by dicyclohexylcarbodiimide or 4-chloro-7-nitro-2,1,3-benzoxadiazole. These observations indicate that 7B3 did not directly interfere with the catalytic sites of ATP or ITP hydrolysis. On the contrary, 7B3 modified the interaction between nucleotide sites and therefore the regulation of the rate of ATP hydrolysis. Indeed, 7B3 changed into a positive cooperativity the negative cooperativity observed when measuring the rate of ATP hydrolysis as a function of ATP concentration. 7B3 also increased the binding of ADP to F1. 7B3 prevented the rapid phase of inactivation of F1 by 5'-p-fluorosulfonylbenzoyladenosine. This phase has been correlated to the binding of 5'-p-fluorosulfonylbenzoyladenosine to regulatory sites (Di Pietro, A., Godinot, C., Martin, J. C., and Gautheron, D. C. (1979) Biochemistry 18, 1738-1745). The inhibition of ATP hydrolysis is concomitant with the binding of 1 mol of IgG or of 2 mol of Fab fragments per mol of F1. However, by further increasing the ratio Fab/F1, only 1 mol of Fab remained bound to F1 without change in inhibition of ATPase activity. All these experiments strongly support the suggestion that F1 conformational changes occurring upon binding of 7B3 to alpha subunit induce a modification of interactions between nucleotide sites. This modification would be consecutive to a change in the normal interaction between the alpha and beta subunits which is required to observe an active rate of ATP hydrolysis or synthesis. In conclusion, the use of this monoclonal antibody demonstrates for the first time in mammalian F1 the role of the conformation of the alpha subunit in the regulation of the ATPase activity.

The rotation of the α subunit of F 1 relative to minor subunits is not involved in ATP synthesis. Evidence given by using an anti-α subunit monoclonal antibody

To test whether ATP synthesis could occur via a mechanism of rotational catalysis in which the α and β subunits of F 1 would rotate with respect to the minor subunits, we have measured the rate of ATP synthesis after binding various masses of antibodies to F 1. If the rotation was an essential feature of the mechanism, the rate of ATP synthesis should be inhibited either completely or proportionately to the load carried by F 1. Bivalent immunoglobulins (IgG) or monovalent Fab fragments of an anti-α monoclonal antibody (7B 3) were bound to F 1 present in electron-transport particles in a ratio of 2 Fab or 2 IgG per F 1. This binding similarly inhibited the rate of ATP synthesis by a maximum of about 50%. When anti-mouse immunoglobulins were added to the F 1-7B 3 (IgG) complex, no significant change in the rate of inhibition was observed. In conclusion, the rate of ATP synthesis was the same when F 1 was loaded with 100 kDa (2 Fab), 300 kDa (2 IgG, 7B 3) or 900 kDa (2 IgG + 4 anti-mouse IgG). It is concluded that the rotation of the α subunits is extremely unlikely to play an essential role in the mechanism of ATP synthesis.

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.

Effects of delipidation on proton translocation and ATPase activity in beef heart electron transport particles.

Delipidation of beef heart electron transport particles with phospholipase A2 has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg2+ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A2 hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and 'specific' leaks through impaired H+-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion. Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17 degrees C (control) to 22 degrees C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A2 rendering the transport particles both leakier and the membranes less fluid than controls.

Fluorescent analogs of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel

N-Cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4) and N-cyclohexyl-N'-(1-pyrenyl)carbodiimide (NCP) are two novel fluorescent analogues of the mitochondrial inhibitor dicyclohexylcarbodiimide (DCCD). Although nonfluorescent in aqueous media, both compounds form fluorescent conjugates with mitochondrial electron transport particles (ETPH) or purified H+-ATPase (F1-F0) vesicles. DCCD prevents the reaction of ETPH with both NCD-4 and NCP. The fluorescent probes are effective inhibitors of ATPase activity and ATP-driven membrane potential, although their reaction rates are considerably slower than that of DCCD. The fluorescence of NCD-4- or NCP-treated H+-ATPase is quenched by hydrophobic spin-label nitroxide derivatives of stearic acid (chi-NS) in the order 16-NS greater than 12-NS greater than 7-NS approximately equal to 5-NS, whereas membrane-impermeant iodide ions have negligible effect. The quenching behavior of 16-NS (the most effective quencher) suggests that a small fraction of labels remain inaccessible to the quencher. It is concluded that the DCCD-binding sites are oriented toward the membrane lipids and are located in the lipid bilayer ca. 18 A from the membrane surface.

Diamide blocks H + conductance in mitochondrial H +-ATPase by oxidizing F B dithiol

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H +-ATPase (F 1–F 0), F 0 of the H +-ATPase from beef heart mitochondria and binding of cadmium ( 109Cd) to the H +-ATPase have been examined in the present paper. When ETPH and purified H +-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistantly inhibited. Diamide also blocks passive H + conductance driven by a K + diffusion potential in the membrane sector, F 0 of H +-ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd 2+ to H +-ATPase, showing competition for the F B dithiol group.

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex.

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.

Interaction of BW755C, a potent inhibitor of lipoxygenase and cyclo-oxygenase, with mitochondrial cytochrome oxidase.

The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytochrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.

841 L-PIPECOLIC ACID (PIP) CATABOLISM IN THE RABBIT: A MODEL FOR PIP METABOLISM IN MAN

Elevated circulating levels of PIP, a putative neurotransmitter, are found in Zellweger syndrome (ZS) and hyperpipecolic acidemia (HPA). As these elevations and the mammalian PIP degradation pathway are poorly understood, we studied L-PIP degradation to α-aminoadipic acid (AAA) in the rabbit. Tissue homogenates converted pure L-[2,3,4,5,6-3H]PIP to 3H-AAA in both rabbit liver and kidney (13.9±9.8 and 34.1±15 pmols/mg prot/hr). Addition of FAD, NAD, and NADP did not raise AAA production and no 3H-AAA was formed in brain homogenates. 3H-AAA identity was confirmed by coelution and re-elution with cold AAA on two paper and one thin layer chromatographic systems. Kidney cortex mitochondria (KCM) formed 3H-AAA from 3H-PIP 14-fold better than tissue homogenates (485±354 pmol/mg prot/hr) and converted 1-2% of the 3H-PIP to 3H-AAA. Addition of 50mM glutamate, a trans-aminase inhibitor, and 1.4mM cold AAA increased AAA recovery 2.7 and 2.8 fold, respectively. Glutamate and cold AAA lowered 3H release from 3H-PIP 45 to 50%, confirming the blockade of 3H-AAA degradation by these compounds. Thus, KCM degrade L-PIP to AAA; the apparent mitochondrial location of this pathway is consistent with its location on the electron transport particle of P. putida, although we cannot totally exclude peroxisomal involvement in mammalian PIP degradation. Further studies on PIP metabolism in rabbit KCM and peroxisomes and in ZS and HPA tissues and fibroblasts are in progress.

Formation of an intramolecular disulfide bond in the mitochondrial adenine nucleotide translocase

Adenine nucleotide translocase in electron transport particles or in H +-ATPase preparation from bovine heart mitochondria is capable of forming both inter- and intramolecular disulfide bridges upon reaction with copper- o-phenanthroline. We have examined the localisation of the intramolecular disulfide bridge in the protein chain by peptide fragmentation methods. The most likely position of the disulfide bridge is between cysteine 159 and 256, but the possibility of the presence of a second disulfide bridge formed between 129 and 256 cannot be ruled out. Our experimental results support the theoretical model proposed [(1982) FEBS Lett. 144, 250-254] for the topography of the translocase and provide a more accurate description of the arrangement of some of the hydrophilic segments in the molecule.

Environment of the sulfhydryl groups in bovine heart mitochondrial H+-ATPase.

Electron transport particles and purified H+-ATPase (F1-F0) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series of N-polymethylene carboxymaleimides (Mal-(CH2)x-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive. N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector F0 was 50% blocked by AM10, slightly blocked by AM2 and N-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 A from the surface are functional in proton translocation through the H+-ATPase.

Identification of the 29,000-dalton protein and its relevance to oligomycin-sensitive 32Pi-ATP exchange in bovine heart electron transport particles.

There have been several reports on the involvement of a 29,000-dalton protein in the regulation of ATP synthesis and 32Pi-ATP exchange (Zimmer, G., Mainka, L., and Heil, B. M. (1982) FEBS Lett. 150, 207-210). The present communication demonstrates that incubation of electron transport particles with 50 microM copper-o-phenanthroline results in reversible loss of 32Pi-ATP exchange but not of oligomycin-sensitive ATPase. Dependence of the inhibition on oxygen, its prevention by EDTA, ATP, or 2-mercaptoethanol, and subsequent restoration of the activity by 2-mercaptoethanol point to a thiol-disulfide interchange as the cause of inhibition. Analysis of copper-o-phenanthroline-treated samples by polyacrylamide gel electrophoresis conducted under nonreducing conditions shows four major changes. There is a decrease in the staining intensity of two bands with molecular weights of 34,000 and 29,000 with concomitant appearance of two new bands with molecular weights of 28,000 and 58,000-60,000. The 34,000-dalton band is tentatively identified as the phosphate transport protein. The 28,000-dalton component is formed by intramolecular and the 58,000-60,000-dalton component by intermolecular cross-linking of the 29,000-dalton protein. Pretreatment of electron transport particles with 2 mM N-ethylmaleimide does not affect 32Pi-ATP exchange or its inhibition by copper-o-phenanthroline but prevents cross-linking of the 34,000- and 29,000-dalton proteins. Evidence is presented to demonstrate that the purified H+-ATPase preparation has a single 29,000-dalton protein, identical to the adenine nucleotide translocase, and that it is not essential for 32Pi-ATP exchange or oligomycin-sensitive ATPase.

Spectral studies on cytochrome d ofAzotobacter vinelandii

Carbon monoxide binding and photodissociation (photolysis) properties of cytochrome d in the membrane electron transport particles ofAzotobacter vinelandii were investigated at cold temperature. The Soret absorption spectrum of cytochrome d was revealed. The evidence supports the thesis that cytochrome d is the major oxidase that carries out NADH oxidative pathway to oxygen at the late-log stage of cell growth inAzotobacter vinelandii.

Nutritional effects on mitochondrial bioenergetics. Alterations in oxidative phosphorylation by rat liver mitochondria.

Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.

Effects of 2-hydroxy-3-undecyl-1,4-naphthoquinone on respiration of electron transport particles and mitochondria: topographical location of the Rieske iron-sulfur protein and the quinone binding site.

2-Hydroxy-3-undecyl-1,4-naphthoquinone is a quinone analogue that inhibits mitochondrial respiration in the cytochrome b-c1 region with an apparent Ki of 2.5 X 10(-7) M. In electron transport particles, it prevents the reduction of cytochrome c1 by succinate but not its oxidation by oxygen and prevents oxidation of cytochrome b but not its reduction by succinate. The analogue increases the amount of steady-state cytochrome b reduction in actively respiring particles. It inhibits oxidant-induced reduction of cytochrome b in the presence of antimycin. Inhibition of succinate oxidase activity in electron transport particles is independent of the pH of the suspending medium while at pH values above 8 with mitochondria, inhibition decreases. Since the apparent pK of the bound quinone is pH 6.6, the pH dependency of the inhibition is likely due to the pK of the Rieske iron-sulfur center (pH 8). The Rieske center and thus the quinone binding site are located on the cytoplasmic face of the inner membrane.

Isolation of totally inverted submitochondrial particles by sonication of beef heart mitochondria.

A novel procedure for isolating totally inverted preparations of submitochondrial particles by sonication of beef heart mitochondria is described. The procedure involves only differential centrifugation in 0.25 M sucrose containing 0.15 M KCl. The submitochondrial particles have 96% of their cytoplasmic face cytochrome c-binding sites sequestered within the particles. Mild sonication exposes cytochrome c-binding sites to the medium. The oligomycin-sensitive ATPase of sonic-derived submitochondrial particles, like that of electron transport particles, is inhibited 98% by exogenous isolated ATPase inhibitor protein. NADH oxidase activity in these particles is inhibited by oligomycin. The respiratory control index (uncoupled rate/oligomycin-inhibited rate) is approximately 3.4 and can be increased by washing the particles with medium containing bovine serum albumin.

Flavin-linked mitochondrial α-glycerophosphate dehydrogenase of Candida utilis

The 150-fold purification of the l-α-glycerophosphate dehydrogenase of Candida utilis electron-transport particles by very mild procedures is described. The active enzyme contains FAD, iron and copper. The function of the metals, if any, is not clear. Its molecular weight is about 5·10 5. The subunit composition is complex and remains unresolved because the enzyme is contaminated with protease(s). The activity of this enzyme is very low in Saccharomyces cerevisiae unless the cells are grown in glycerol. The NAD-dependent cytoplasmic α-glycerophosphate dehydrogenase is present in C. utilis but could not be demonstrated in glucose-grown S. cerevisiae.

Differential exposure of components of cytochrome b-c1 region in beef heart mitochondria and electron transport particles.

The reduction of cyctochromes c + c1 by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochrome c-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% of c + c1 in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromes c + c1 (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromes c + c1. Addition of ferrocyanide to intact cytochrome c-depleted mitochondria does not reduce cytochrome c1; treatment with N,N,N',N'-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromes c + c1. The Km value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochrome c1 is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochrome c1 is exposed to the matrix face and cytochrome c is exposed to the cytoplasmic face. No redox center other than cytochrome c in the segment between the antimycin site and cytochrome c is exposed on the C-side.