A conversation with John E. Walker interviewed by K. Noelle Gracy.
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ATP Synthetase Complexes↗
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ATP synthase: constrained stoichiometry of the transmembrane rotor.
Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.
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Thyroid hormone increases transcription of GA-binding protein/nuclear respiratory factor-2 alpha-subunit in rat liver.
Thyroid hormone (TH) regulates mitochondrial respiratory rate by activating coordinated transcription in the nucleus and mitochondria. Whereas TH activates transcription of mitochondrial genes directly, the activation of nuclear-encoded mitochondrial genes is probably executed by indirect unknown mechanisms. Nuclear respiratory factors (NRF)-1 and GA-binding protein (BP)/NRF-2 may function as transacting genes, but regulation of these genes by TH is not demonstrated. We show that TH administration to hypothyroid rats promptly increases GABP/NRF-2 alpha-subunit mRNA levels in the liver, without significant changes in beta, gamma subunits. In run-on and time-course experiments, the transcription rate and protein levels increased three-fold in response to TH, indicating GABP/NRF-2 transcriptional regulation. The results also support the notion that ATP synthase beta-subunit is regulated by TH through the indirect activation of GABP/NRF-2.
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ATP synthase: a tentative structural model.
Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer.
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Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages.
Interleukin-8 (IL-8) is a chemotactic factor for T-lymphocytes and smooth muscle cells and may therefore have an important effect in atherogenesis. It is secreted from oxysterol-containing foam cells which have been found in hypoxic zones in atherosclerotic plaques. The aim of this study was to investigate the effect of hypoxia on the secretion of IL-8 by oxysterol-stimulated macrophages. Hypoxia enhances 25-hydroxycholesterol (25-OH-chol)-induced IL-8 secretion in human monocyte-derived macrophages. The effect is most pronounced when macrophages are incubated with low concentrations of 25-OH-chol. Both 25-OH-chol and hypoxia increases the intracellular level of the signalling molecule hydrogen peroxide (H(2)O(2)). This event coincided with an enhanced binding of the transcription factor c-jun to the IL-8 gene promoter and an increased IL-8 mRNA expression in hypoxic macrophages. These observations suggest that similar intracellular signalling pathways are used for both 25-OH-chol-induced IL-8 expression and hypoxia-induced IL-8 expression. Thus, hypoxia in atherosclerotic plaques may increase the secretion of IL-8 from oxysterol-containing foam cells, which subsequently may accelerate the progression of atherosclerosis.
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Bacterial Na+ - or H+ -coupled ATP synthases operating at low electrochemical potential.
In certain strictly anaerobic bacteria, the energy for growth is derived entirely from a decarboxylation reaction. A prominent example is Propionigenium modestum, which converts the free energy of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA (DeltaG degrees =-20.6 kJ/mol) into an electrochemical Na(+) ion gradient across the membrane. This energy source is used as a driving force for ATP synthesis by a Na(+)-translocating F(1)F(0) ATP synthase. According to bioenergetic considerations, approximately four decarboxylation events are necessary to support the synthesis of one ATP. This unique feature of using Na(+) instead of H(+) as the coupling ion has made this ATP synthase the paradigm to study the ion pathway across the membrane and its relationship to rotational catalysis. The membrane potential (Deltapsi) is the key driving force to convert ion translocation through the F(0) motor components into torque. The resulting rotation elicits conformational changes at the catalytic sites of the peripheral F(1) domain which are instrumental for ATP synthesis. Alkaliphilic bacteria also face the challenge of synthesizing ATP at a low electrochemical potential, but for entirely different reasons. Here, the low potential is not the result of insufficient energy input from substrate degradation, but of an inverse pH gradient. This is a consequence of the high environmental pH where these bacteria grow and the necessity to keep the intracellular pH in the neutral range. In spite of this unfavorable bioenergetic condition, ATP synthesis in alkaliphilic bacteria is coupled to the proton motive force (DeltamuH(+)) and not to the much higher sodium motive force (DeltamuNa(+)). A peculiar feature of the ATP synthases of alkaliphiles is the specific inhibition of their ATP hydrolysis activity. This inhibition appears to be an essential strategy for survival at high external pH: if the enzyme were to operate as an ATPase, protons would be pumped outwards to counteract the low DeltamuH(+), thus wasting valuable ATP and compromising acidification of the cytoplasm at alkaline pH.
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Identification of differentially expressed genes in human trophoblast cells by differential-display RT-PCR.
Molecular mechanisms controlling human trophoblast invasiveness are still poorly understood. In the present investigation, mRNA patterns of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared by differential-display reverse transcriptase polymerase chain reaction (DDRT-PCR) revealing differential expression of numerous genes. Of 18 differentially expressed DDRT-PCR products analysed, 11 were unknown, four showed homologies with expressed sequence tag sequences, and three others were homologous to integrin-beta1, ATP-synthetase U6 (both showing higher expression in first trimester) or to aldose-reductase (higher expression at term), respectively. One of the unknown transcripts (PBK1, accession number: AJ007398) was cloned from a first trimester placenta cDNA library and was characterized. The 1908-bp gene fragment contains an open reading frame of 1551 bp and an Alu-sequence in the 3' non-coding region. According to Northern blot analysis on JAr choriocarcinoma cells, the fragment is close to full-length cDNA. By in situ hybridization, PBK1 was detected only in first trimester but not term placentae in the proximal parts of cell islands and in closely adjacent villous cytotrophoblast. This expression pattern suggests that the newly identified molecule, PBK1, could be involved in the regulation of proliferation/ differentiation and potentially in invasion of trophoblast cells.
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Severe energy impairment consequent to inactivation of mitochondrial ATP synthase as an early event in cell death: a mechanism for the selective sensitivity to H2O2 of differentiating erythroleukemia cells.
Irreversible damage to Friend's erythroleukemia cells was caused by induction of endogenous heme biosynthesis with the differentiating agent N,N'-hexamethylene bisacetamide followed by a 30-min exposure to 0.25 mM H2O2. Early irreversible ATP depletion was observed concomitant with oxidative inactivation of the mitochondrial ATP synthase. Cell proliferative capacity was also impaired within 2 h of the treatment, and progressive delayed cell lethality, starting 2 h after the insults, was also found. Based on the prevention provided by specific antioxidants and on the absence of malodialdehyde production, all the effects were ascribed to the oxidant action of .OH radicals, or closely related species, generated through iron-catalyzed reactions of H2O2, which apparently caused site-directed oxidative modifications of iron-binding proteins, in particular mitochondrial ATP synthase, rather than peroxidation of membrane lipids. Similar effects were mimicked even in the parental cell line when oligomycin was used to inhibit selectively mitochondrial ATP synthase activity, thereby lowering the enzyme activity to a level similar to that found in H2O2-damaged differentiating cells. Hence, induction of erythroid differentiation makes the mitochondrial ATP synthase a major target of H2O2 by enhancing the availability of redox-active iron in the local environment of the enzyme. Subsequent oxidative inactivation of the mitochondrial ATP synthase, resulting in severe energy impairment, leads to loss of cell growth capacity. Erythroleukemia cells may serve as a model system for the combination of two selective properties: (1) the capacity for carrying out efficient heme synthesis and/or for undergoing iron overload-like state; and (2) subsequent enhanced sensitivity to reactive oxygen species generators. Early severe mitochondrial dysfunction and energy impairment may be a major part of the mechanism of the sensitivity.
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The synthesis and antibacterial activity of totarol derivatives. Part 3: Modification of ring-B.
Ring-B derivatization of totarol (1) afforded the series of compounds 2-22 which were screened in vitro against: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the antibacterial activity of totatol against the first three of these organisms (all gram-positive), but none was more active. The gram-negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in isolated mitochondria at 50 microM.
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NMR studies of enzymatic rates in vitro and in vivo by magnetization transfer.
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Differential effects of chilling-induced photooxidation on the redox regulation of photosynthetic enzymes.
Photosynthesis in plant species that are evolutionarily adapted for growth in warm climates is highly sensitive to illumination under cool conditions. Although it is well documented that illumination of these sensitive species under cool conditions results in the photosynthetic production of reactive oxygen molecules, the underlying mechanism for the inhibition of photosynthesis remains uncertain. Determinations of chloroplast fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase activity showed that the light-dependent, reductive activation of these key carbon reduction cycle enzymes was substantially inhibited in tomato (Lycopersicon esculentum) following illumination at 4 degrees C. However, other chloroplast enzymes also dependent on thioredoxin-mediated reductive activation were largely unaffected. We performed equilibrium redox titrations to investigate the thermodynamics of the thiol/disulfide exchange between thioredoxin f and the regulatory sulfhydryl groups of fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde phosphate dehydrogenase, and the chloroplast ATPsynthase. We determined that the redox midpoint potentials for the regulatory sulfhydryl groups of the various enzymes spanned a broad range ( approximately 50 mV at pH 7. 9). The electron-sharing equilibria among thioredoxin f and its target enzymes largely explained the differential effects of photooxidation induced at low temperature on thioredoxin-mediated activation of chloroplast enzymes in tomato. These results not only provide a plausible mechanism for the low-temperature-induced inhibition of photosynthesis in this important group of plants, but also provide a quantitative basis to evaluate the influence of thioredoxin/target enzyme electron-sharing equilibria on the differential activation and deactivation kinetics of thioredoxin-regulated chloroplast enzymes.
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Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases.
Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.
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Estimation of H+ to adenosine 5'-triphosphate stoichiometry of Escherichia coli ATP synthase using 31P NMR.
High-field 31P NMR techniques have been used to measure transmembrane delta pH in wild-type, unc A, and hem A mutants of Escherichia coli. delta psi was measured by distribution methods with radioactive tetraphenylphosphonium bromide and 86Rb+ ions as the probes, while intracellular ATP, ADP, and inorganic phosphate concentrations were determined from the 31P NMR spectra. delta G'p and the stoichiometry for ATP synthesis [delta G'p/(F delta p)] were then calculated. The stoichiometry of the ATP synthase was found to vary as a function of the cellular metabolic state. In nongrowing, wild-type cells delta p was 192 +/- 16 mV with succinate as the substrate and saturating oxygen tension. With limiting oxygen (congruent to microM oxygen), delta p was 125 +/- 14 mV. Nucleoside triphosphate synthesis was observed in both cases. The H+/ATP stoichiometry varied from 2.15 +/- 0.35 under aerobic conditions to 3.6 +/- 0.8 at low oxygen tension. delta p for unc A cells was 140 +/- 14 mV with glucose as the substrate (greater than 2.5 microM oxygen) and for hem A mutants was 115 +/- 10 mV. The bulk phase potentials in oxygen-limited, wild-type cells and in respiratory deficient (hem A) cells are comparable, but in the former the ATPase is poised for synthesis while in the latter it generates delta p. The data support a role for localized interactions between the redox and the ATPase sites.
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Kinetic control of mitochondrial ATP synthesis.
In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
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Coupling factor for photophosphorylation labeled with eosin isothiocyanate: activity, size, and shape in solution.
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Mitoproteome plasticity of rat brown adipocytes in response to cold acclimation.
Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.
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Reversible dysregulation of the mitochondrial ATP synthase in skin fibroblasts from children with vitamin B12-dependent methylmalonic acidaemia.
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Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.
The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP<-->Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.
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