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PubMed1982

Unifying concept for the coupling between ion pumping and ATP hydrolysis or synthesis.

A mechanism is proposed for the coupling between ion transport and enzyme catalysis. The basic concept is that enzymes associated with transport exist in two possible conformations. Each conformation has the potential of catalyzing the enzymatic reaction, and pumping is associated with the conversion of one conformational form to the other. The conformational transition is triggered by the kinetic blockage of specific mechanistic steps for each conformation. Such blockages can cause a cycling between the two conformations concomitant with catalysis. This mechanistic concept is consistent with a variety of results obtained with the Na+,K+-ATPase, the Ca2+,Mg2+-ATPase, and ATP synthesizing enzymes (coupling factors).

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PubMed1982

On the enzymic mechanism of oxidative phosphorylation.

Oxidative phosphorylation, like substrate-level phosphorylation, involves oxidative conversion of inorganic phosphate to a reactive species followed by interaction of this species with enzyme-bound ADP to form enzyme-bound ATP. The reactive species in a phosphoryl ester in substrate-level phosphorylation and phosphonium ion of orthophosphate in oxidative phosphorylation. The coupled synthesis is mediated by a combination of two classical enzymes in substrate-level phosphorylation and by a set of energy-coupled enzymes in oxidative phosphorylation. The full range of experimental evidence supporting this proposed enzymic mechanism of oxidative phosphorylation is presented as well as the rationalization of phenomena that hitherto have eluded explanation.

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PubMed1998

The 2.8-A structure of rat liver F1-ATPase: configuration of a critical intermediate in ATP synthesis/hydrolysis.

During mitochondrial ATP synthesis, F1-ATPase-the portion of the ATP synthase that contains the catalytic and regulatory nucleotide binding sites-undergoes a series of concerted conformational changes that couple proton translocation to the synthesis of the high levels of ATP required for cellular function. In the structure of the rat liver F1-ATPase, determined to 2.8-A resolution in the presence of physiological concentrations of nucleotides, all three beta subunits contain bound nucleotide and adopt similar conformations. This structure provides the missing configuration of F1 necessary to define all intermediates in the reaction pathway. Incorporation of this structure suggests a mechanism of ATP synthesis/hydrolysis in which configurations of the enzyme with three bound nucleotides play an essential role.

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PubMed1997

Cellular respiration during hypoxia. Role of cytochrome oxidase as the oxygen sensor in hepatocytes.

We previously reported that hepatocytes exhibit a reversible suppression of respiration during prolonged hypoxia (PO2 = 20 torr for 3-5 h). Also, isolated bovine heart cytochrome c oxidase undergoes a reversible decrease in apparent Vmax when incubated under similar conditions. This study sought to link the hypoxia-induced changes in cytochrome oxidase to the inhibition of respiration seen in intact cells. Hepatocytes incubated at PO2 = 20 torr exhibited decreases in respiration and increases in [NAD(P)H] after 2-3 h that were reversed upon reoxygenation (PO2 = 100 torr). Respiration during hypoxia was also inhibited when N,N,N',N'-tetramethyl-p-phenylenediamine (0.5 mM) and ascorbate (5 mM) were used to reduce cytochrome c, suggesting that cytochrome oxidase was partially inhibited. Similarly, liver submitochondrial particles revealed a 44% decrease in the apparent Vmax of cytochrome oxidase after hypoxic incubation. In hepatocytes loaded with tetramethylrhodamine ethyl ester (10 nM) to quantify mitochondrial membrane potential, acute hypoxia (<30 min) produced no change in fluorescence, consistent with the absence of an acute change in respiration. However, fluorescence increased during acute reoxygenation after prolonged hypoxia, suggesting an increase in potential. The control exhibited by NADH over mitochondrial respiration was not altered during hypoxia. Thus, changes in the Vmax of cytochrome oxidase during prolonged hypoxia correlate with the changes in respiration and mitochondrial potential. This suggests that the oxidase functions as an oxygen sensor in the intact hepatocyte.

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PubMed1997

Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis.

The participation of the amino acid beta83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta83 was changed from Glu to Asp (betaE83D) and to Lys (betaE83K), and the highly conserved tetrapeptide betaT82-E83-G84-L85 (DeltaTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15DeltaatpB and FUD50 with the mutated atpB genes. The transformants containing the betaE83D and betaE83K mutant genes grew well photoautotrophically. The DeltaTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped DeltapH with thylakoids isolated from cw15 and the two mutants, betaE83D and betaE83K, were similar. However, only the phosphorylation activity of the mutant betaE83D was inhibited by tentoxin with 50% inhibition attained at 4 microM. These results confirm that amino acid beta83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, betaE83D, and betaE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the betaE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the betaE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the betaE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.

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PubMed2000

Differential regulation of exonic regulatory elements for muscle-specific alternative splicing during myogenesis and cardiogenesis.

Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

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PubMed2004

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme.

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.

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PubMed2002

Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function.

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.

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PubMed2002

A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning, localization, and putative function.

Mitochondrial AAA metalloproteases play a fundamental role in mitochondrial biogenesis and function. They have been identified in yeast and animals but not yet in plants. This work describes the isolation and sequence analysis of the full-length cDNA from the pea (Pisum sativum) with significant homology to the yeast matrix AAA (m-AAA) protease. The product of this clone was imported into isolated pea mitochondria where it was processed to its mature form (PsFtsH). We have shown that the central region of PsFtsH containing the chaperone domain is exposed to the matrix space. Furthermore, we have demonstrated that the pea protease can complement respiration deficiency in the yta10 and/or yta12 null yeast mutants, indicating that the plant protein can compensate for the loss of at least some of the important m-AAA functions in yeast. Based on biochemical experiments using isolated pea mitochondria, we propose that PsFtsH-like m-AAA is involved in the accumulation of the subunit 9 of the ATP synthase in the mitochondrial membrane.

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PubMed2004

Progression and specificity of protein oxidation in the life cycle of Arabidopsis thaliana.

Protein carbonylation is an irreversible oxidative process leading to a loss of function of the modified proteins, and in a variety of model systems, including worms, flies, and mammals, carbonyl levels gradually increase with age. In contrast, we report here that in Arabidopsis thaliana an initial increase in protein oxidation during the first 20 days of the life cycle of the plant is followed by a drastic reduction in protein carbonyls prior to bolting and flower development. Protein carbonylation prior to the transition to flowering targets specific proteins such as Hsp70, ATP synthases, the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and proteins involved in light harvesting/energy transfer and the C2 oxidative photosynthetic carbon cycle. The precipitous fall in protein carbonyl levels is due to the specific reduction in the levels of oxidized proteins rather than an overall loss of chlorophyll and Rubisco associated with the senescence syndrome. The results are discussed in light of contemporary theories of aging in animals.

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PubMed2006

Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons.

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.

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PubMed2000

Autophagy in embryonic erythroid cells: its role in maturation.

Yolk sac-derived embryonic erythroid cells differentiate synchronously in the peripheral blood of Syrian hamster. The stage of differentiation on day 10 of gestation is equivalent to polychromatophilic erythroblast stage and that on day 13 is equivalent to the reticulocyte stage in adult animals. The cytoplasm of embryonic erythroid cells became scant and devoid of most organelles on day 12 of gestation. In addition, there were very few non-erythroid cells in circulation before day 13. Thus the embryonic erythroid cells serve a pure and synchronous system to study the mechanisms of terminal differentiation. The number of mitochondria in the embryonic erythroid cells decreased to about 10% of the initial number during the period between day 10 and day 12 of gestation. In contrast, the frequency of autophagy of mitochondria increased 4.6-fold in the same period. The cytochrome c content of the cell decreased as the mitochondria became extinct. However, release of cytochrome c into the cytoplasm was not detectable through day 10-13 of gestation, suggesting that the mitochondria were digested within a closed compartment. Decomposed mitochondria and ferritin particles were detected in lysosomes by electron microscopy on and after day 12 of gestation, which also suggested digestion in a closed compartment. Mitochondrial ATP synthase subunit c, which is known to be a protease-refractory protein, was retained in the cells even after the disappearance of mitochondria, indicating that most of the mitochondria were not extruded from the cells. The digestion of mitochondria in autolysosomes may allow the cells to escape from rapid apoptotic cell death through concomitant removal of mitochondrial death-promoting factors such as cytochrome c.

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PubMed2003

Rotary movements within the ATP synthase do not constitute an obligatory element of the catalytic mechanism.

After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism. On the other hand, the experimental evidence in favor of a two-sites catalytic mechanism, derived from various approaches and not compatible with a three-sites rotary mechanism, appear to be very solid. Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation.

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PubMed1998

Variations in gene expression and genomic stability of human hepatoma cells integrated with hepatitis B virus DNA.

The association of hepatitis B virus (HBV) infection with human hepatoma is well established. However, no consensus regarding the etiology of hepatocellular carcinoma was elucidated. In this paper, the genomic stability and gene expression of HBV DNA-integrated and non-integrated hepatoma cells by Transcript Profile (TP)-PCR and RT-PCR are characterized. The additional DNA bands generated from TP-PCR of HBV integrated genomes were not correlated with the sequence of HBV, suggesting that the variations may result from genomic instability of the host cells. Moreover, differential genes expressed in HBV DNA-integrated cells were sequenced. A cDNA generated from the integrated cells exhibited 99.3% homology with the sequences of ATP synthase 6 and cytochrome C oxidase III, but the sequences were abnormally linked together. Since HBV infection may alter the energy metabolism of the cell, the results suggest that the integration may cause mitochondriae defects in the ATP synthase 6 and cytochrome C oxidase III genes.

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PubMed2000

Identification of four genes of the Brucella melitensis ATP synthase operon F0 sector: relationship with the Rhodospirillaceae family.

We have determined the nucleotide sequence of a cloned DNA fragment from the human and animal pathogen Brucella melitensis. Four genes were identified from a 4069 bp fragment, corresponding to the B. melitensis a, c, b', and b subunits of the ATP synthase F0 sector operon. A duplicated and divergent copy of the b-subunit gene was observed. This feature has been found only in photosynthetic bacteria and chloroplasts. In addition, the gene cluster was separated from the F1 sector, a characteristic described only for the Rhodospirillaceae family.

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PubMed1981

The atp operon: nucleotide sequence of the promoter and the genes for the membrane proteins, and the delta subunit of Escherichia coli ATP-synthase.

The nucleotide sequence of the promoter region and the first five genes of the atp (or unc) operon of Escherichia coli has been determined. The first proposed gene in the operon contains four AUA codons and may be poorly expressed; it encodes a basic but yet hydrophobic protein which could function as a pilot protein for assembly of ATP-synthase. The three genes that follow are structural genes for proteins comprising the proton channel of the enzyme. The fifth gene codes for the delta-subunit of F(1)-ATPase.

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PubMed1981

The atp operon: nucleotide sequence of the region encoding the alpha-subunit of Escherichia coli ATP-synthase.

Part of the atp (or unc) operon encoding the alpha, beta, gamma, delta, and epsilon subunits of Escherichia coli ATP-synthase has been cloned into the plasmid pACYC 184. The DNA coding for the largest of these proteins, the alphas subunit, has been sequenced by cloning into the bacteriophage M13 and sequencing with dideoxy nucleotide chain terminators. It comprises 1539 nucleotides corresponding to a protein of 513 amino acids.

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