Modifications of myosin caused by adenosine-triphosphate,adenosinediphosphate, and pyrophosphate

Riassunto In presenza di ATP o ADP o pirofosfato, in concentrazioni da 10^–2 a 10⁴ M, la solubilità della miosina aumenta notevolmente a bassa forza ionica. Il legame che si forma fra la miosina e i polifosfati è tale che solo una dialisi prolungata può allontanarli interamente.     

The effect of temperature on Ca2+ uptake and Ca2+-activated ATP hydrolysis by cardiac sarcoplasmic reticulum

Zusammenfassung Der Ca²⁺-Transport und die Ca²⁺-aktivierte ATP Hydrolyse (ATP extra Spaltung) durch Membranen des cardialen sarkoplasmatischen Retikulums zeigen die gleiche Temperaturabhängigkeit. Die Aktivierungsenergie der Ca²⁺-Aufnahme und der ATP extra Spaltung, gemessen bei Anwesenheit von Oxalat, beträgt 16.65±0.87 und 17.93±0.49 Kcal/Mol^–1.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis

A single cDNA of cytochrome c oxidase subunit VIa was characterised from liver, heart and the thermogenic organ of the partially endotherm tuna fish. The amino acid sequence revealed high identity with subunit VIa from carp and trout, but low identity to subunits VIaL (liver type) and VIaH (heart type) of mammalian cytochrome c oxidase. In reconstituted cytochrome c oxidase from bovine heart, the H ⁺/e⁻ stoichiometry is decreased from 1.0 to 0.5 at high intraliposomal ATP/ADP ratios via exchange of bound ADP by ATP at the matrix domain of the transmembraneous subunit VIaH. Reconstituted cytochrome c oxidase from bovine liver and kidney, containing subunit VIaL, revealed H ⁺/e⁻ ratios below 0.5, independent of the ATP/ADP ratio. The results suggest the evolution of three types of subunit VIa. Subunits VIaH and VIaL are postulated to participate in mammalian thermogenesis. Received 3 May 1999; received after revision 10 June 1999; accepted 29 June 1999     

Allometry of mammalian cellular oxygen consumption

In the 1930s, Max Kleiber and Samuel Brody established that the interspecies correlation between mammalian body mass and metabolic rate (αM^0.75) cannot be explained (solely) by whole body surface area (αM^0.66) to volume ratios. Metabolic considerations must also be taken into account. Decreases in the proportion of visceral organ mass to whole body mass can account for some of the whole body metabolic differences. However, superimposed upon these anatomical differences, the metabolism of tissues and cells has been demonstrated to decrease with increasing body mass. These decreases in oxygen consumption rates (with increasing body mass) in cells and tissues can be explained by a decrease in ATP turnover and mitochondrial density and an increase in mitochondrial functional efficiency (decrease in proton leak). The majority of the proton leak differences reflect differences in mitochondrial inner membrane surface area. Indeed, liver metabolism correlates directly with liver mitochondrial inner membrane surface area. Apart from being a significant contributor (~25 %) to basal metabolism, mitochondrial proton leak is a major factor determining the differences in basal metabolism between mammals of different body mass. Received 31 May 2000; received after revision 2 October 2000; accepted 14 November 2000     

Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria

Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca²⁺ and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 (_dPC12), _dSH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in _dPC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca²⁺ and ΔΨm. The response to Baf is regulated by cytosolic Ca²⁺ fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.     

Effects of known and suspected neurotransmitter substances and of some nucleotides on isolated mast cells

Résumé Des suspensions de mastocytes péritoneaux isolées de rat furent incubées avec 10 substances différentes, connues ou presumées être des neurotransmitteurs. Seulement l'adenos ine triphosphate (ATP) aux concentrations supérieures à 2.64×10^–6 M causèrent la dégranulation des mastocytes. L'ATP cause également la dégranulation des mastocytes dans le mésentère. L'action de l'ATP peut être responsible de la dégranulation des mastocytes cutanées observée après la stimulation antidromique des nerfs sensorieux, alors que de l'ATP est libéré dans la peau.     

Does an inhibitor of mitochondrial adenylate kinase also affect oxidative phosphorylation?

Summary Adenylate kinase activity of intact mitochondria is strongly inhibited by Ap₅A, i.e.p ¹,p ⁵-Di (adenosine-5-) pentaphosphate, whereas oxidative phosphorylation is not affected. Therefore, Ap₅A is a useful tool to distinguish between oxidative and non oxidative ATP generating reactions.Acknowledgment. The generous support of Prof. Dr.Walther Lamprecht is gratefully acknowledged. J. L. thanks the Stipendienfonds des Verbandes der Chemische Industrie for a scholarship.     

Amin-transport hypothalamischer Vesikel

Summary Noradrenaline storing vesicles were isolated from the pig hypothalamus and incubated at 0° and 37 °C. In the presence of ATP the uptake of noradrenaline is activated by Mg⁺⁺ but not by Ca⁺⁺. At 37 °C the passive transport of noradrenaline is practically accomplished within 1 min, the active one within 6 min.

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Ausgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Drug interactions in intestinal absorption of3H-digitoxin in rats

Summary The absorption of³H-digitoxin from perfused rat small intestine was inhibited by probenecid (1.0×10^–2 M), ethacrynic acid (0.5×10^–3 M), and mersalyl (8.0×10^–3 M) indicating that digitoxin absorption is at least partly an active process.     

Beeinflussung der ATPase aminspeichernder Granula von Nebennierenmark und Milznerven durch Thallium

Summary ATPases of the amine storing granules from bovine adrenal medulla and splenic nerves are inhibited by Tl⁺⁺⁺ 3×10^–5 and 5×10^–6 M, respectively. Tl⁺ up to 10^–3 M is ineffective. By T1⁺⁺⁺ in concentrations of 10^–4 M or more, proteins are precipitated, so that the enzyme inhibiton by these concentrations is unspecific. If T1⁺ is oxidized to Tl⁺⁺⁺ in the organism, the inhibition of granular ATPase may be responsible for the alterations of the catecholamine metabolism observed in thallium intoxication.     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

Net ATP synthesis by running the red cell calcium pump backwards

Summary Ca²⁺ loaded inside-out vesicles from human red blood cells, yielding Ca²⁺ into a Ca²⁺ free medium with 4 mM EGTA, 2 mM ADP and 10 mM phosphate, produced an excess of 14.9 pmoles · min^–1 · (mg protein)^–1 of ATP compared to controls in which the transmembrane Ca²⁺ gradient was abolished by the ionophore A 23 187.We are obliged to Dr H. Fey and Miss H. Pfister (Veterinarybacteriological Institute Bern) and Dr H. Porzig (Pharmacological Institute Bern) for help and advice.     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Redundant and unique roles of coronin proteins in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Dictyostelium discoideum harbors a short (CRN12) and a long coronin (CRN7) composed of one and two beta-propellers, respectively. They are primarily present in the cell cortex and cells lacking CRN12 (corA ⁻) or CRN7 (corB ⁻) have defects in actin driven processes. We compared the characteristics of a mutant cell line (corA ⁻ /corB ⁻) lacking CRN12 and CRN7 with the single mutants focusing on cytokinesis, phagocytosis, chemotaxis and development. Cytokinesis, uptake of small particles, and developmental defects were not enhanced in the corA ⁻ /corB ⁻ strain as compared to the single mutants, whereas motility and phagocytosis of yeast particles were more severely impaired. It appears that although both proteins affect the same processes they do not act in a redundant manner. Rather, they often act antagonistically, which is in accordance with their proposed roles in the actin cytoskeleton where CRN12 acts in actin disassembly whereas CRN7 stabilizes actin filaments and protects them from disassembly.     

Binding of [3H]GABA and [3H]muscimol to subcellular particles of a neurone-enriched culture of mouse brain

Summary Binding of [³H]GABA and [³H]muscimol, indicative of GABA-receptors, has been demonstrated in a neurone-enriched culture of embryonic mouse brain using a ligand-binding technique. Evidence is provided for the existence of different populations of GABA-receptors.This study was supported by C.N.R.S., ATP N^o 3356 and by Commissariat à l'Energie Atomique, Departement of Biologie. Thanks are due to Mlle Marie-Anne Frenkel for her excellent technical assistance.     

Effect of strychnine on the electroretinogram of the isolated rabbit retina

Zusammenfassung Strychnin in einer Konzentration von 3×10^–6 bis 3,5×10^–5 M/l verursacht eine vorübergehende Vergrösserung derb-Welle des ERG der isolierten Kaninchennetzhaut, in einer Konzentration von 7×10^–5 bis 7×10^–4 M/l verkleinert es dieb-Welle. Aus den Ergebnissen wird geschlossen, dass die Substanz zumindest teilweise schon auf die Schicht des zweiten Neurons wirkt.     

Interaction du 2,4-dichlorophénol et de l'eau oxygénée dans la destruction enzymatique de l'acideβ-indolylacétique

Summary DCP increases IAA destruction by bothLens andPhaseolus root breis. H₂O₂ inhibits catabolism byLens extracts but activates it whenPhaseolus is used, mainly when roots are cultivated in the dark and contain inhibitors of IAA destruction. DCP 1·10^–3 M and H₂O₂ 1·10^–4 or 1·10^–3 volume forLens and DCP 1·10^–4 M and H₂O₂ 1·10^–3 volume forPhaseolus nullify auxin catabolism.     

Dynamic laser light scattering studies of the effects of pyrophosphate on cyclic motions of cross-bridges in isolated thick myofilaments fromLimulus striated muscle

Pyrophosphate (PP_i) is a non-hydrolyzable ATP analogue known to affect the binding between myosin heads and actin. By using a dynamic laser light scattering method, we have shown that 1–2 mM PP_i enhances the increase in values induced by Ca²⁺ in isolated thick myofilaments fromLimulus striated muscle. However, similar treatment has no effect on the values of filaments suspended in either relaxing solution or ATP-free solution. is the average linewidth of the photoelectron count autocorrelation function of the light scattered. PP_i had no effect on the increase of values by Sr²⁺ but it substantially increased the values of the thick myofilaments suspended in Ba²⁺-substituted Ca²⁺ activating solution. The results show that PP_i also affects the energy-requiring cyclic cross-bridge motions.     

Influence of cyclic nucleotides on protein synthesis in vascular smooth muscle

Summary The incorporation of leucice-¹⁴C into protein in bovine mesenteric arteries was augmented by cyclic GMP (10^–3 M) and decreased by cyclic AMP (10^–3 M). There was no effect of 5 AMP (10^–3 M). The phosphodiesterase inhibiting drugs theophylline (10^–3 M) and papaverine (5×10^–5 g/ml) both decreased the leucine-¹⁴C incorporation.We are indebted to Mrs.Lena Burlin for hear assistance. Finacial support has been provided by the Swedish State Medical Research Council (No. 04X-101X-4498).