Is ATP synthesized by a vacuolar-ATPase in the extremely halophilic bacteria?

The proton-dependent synthesis of ATP was demonstrated in representative members of the generaHalobacterium, Haloarcula, andHaloferax. In all cases, synthesis was not inhibited by nitrate or N-ethylmaleimide, inhibitors of the vacuolar-like ATPase found in Archaea, but was affected by azide, an inhibitor of F₀F₁-ATP syntheses. These observations extend the earlier observations withHalobacterium saccharovorum and suggest that ATP synthesis in these organisms is brought about by an F₀F₁-APT synthase.     

Recognition of very low concentrations of ATP byGlossina tachinoides Westwood

Summary Like many other blood feeders,Glossina tachinoides is stimulated to gorge by the presence of ATP in its diet. A concentration of 1.3×10^–8 M ATP induces 50% feeding. The ability ofG. tachinoides to detect ATP is the highest recorded so far among insects.     

Proteases and protein degradation inEscherichia coli

InE. coli, protein degradation plays important roles in regulating the levels of specific proteins and in eliminating damaged or abnormal proteins.E. coli possess a very large number of proteolytic enzymes distributed in the cytoplasm, the inner membrane, and the periplasm, but, with few exceptions, the physiological functions of these proteases are not known. More than 90% of the protein degradation occurring in the cytoplasm is energy-dependent, but the activities of mostE. coli proteases in vitro are not energy-dependent. Two ATP-dependent proteases, Lon and Clp, are responsible for 70–80% of the energy-dependent degradation of proteins in vivo. In vitro studies with Lon and Clp indicate that both proteases directly interact with substrates for degradation. ATP functions as an allosteric effector promoting an active conformation of the proteases, and ATP hydrolysis is required for rapid catalytic turnover of peptide bond cleavage in proteins. Lon and Clp show virtually no homology at the amino acid level, and thus it appears that at least two families of ATP-dependent proteases have evolved independently.     

About the photostimulation of ATP production in the integument of insects

Summary The photostimulation of ATP production in the integument ofPieris brassicae has been reexamined, using 2 different methods for ATP estimation. As could be demonstrated with the luciferin-luciferase assay, the so-called photostimulation is an artefact which is due to inappropriate utilization of the Boehringer kit assay.     

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H₂O₂, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase α chain is probably tyrosine-phosphorylated, but demonstrated that the β chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.Received 7 January 2005; received after revision 19 April 2005; accepted 22 April 2005     

Adénosine triphosphatase et pyrophosphatase des chloroplastes

Summary ATP hydrolysis and inorganic pyrophosphate hydrolysis in chloroplasts of spinach leaves are characterized by a different pH optimum, a different sensitivity to magnesium ions, top-chloromercuribenzoate and to ageing. It is concluded that ATP and inorganic pyrophosphate are likely hydrolyzed by two different enzymes in chloroplasts.     

Degradation of type I collagen fibrils synthesized by human dental pulp cells in explant culture exposed toActinomyces viscosus. Electron microscope immunotyping

Summary Actinomyces viscosus Be 66, added to pulpal cells in culture, does not cause apparent cellular damage. The extracellular matrix consists of altered collagen fibrils and thin filaments, immunochemically identified as type I collagen. They probably represent the first steps of collagen degradation.This work was supported by INSERM (ATP: 77-85) and CNRS (RCP: 533).     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

Cell surface adenylate kinase activity regulates the F1-ATPase/P2Y13-mediated HDL endocytosis pathway on human hepatocytes

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase and nucleoside diphosphokinase activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F₁-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation. Received 13 July 2006; received after revision 29 August 2006; accepted 19 September 2006     

Phosphatases ofMycobacterium 607

Zusammenfassung Die Phosphatase(n)-Aktivität der zellfreien Extrakte vonMycobacterium 607 wurde untersucht und Pyrophosphat, ATP, ADP und Fructose-1,6-diphosphat hydrolisiert. Eigene Eigenschaften der Phosphatasen wurden studiert. Auch wurde nachgewiesen, dass die Hydrolyse von ATP und ADP durch mindestens zwei verschiedene Enzyme hervorzurufen ist.

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Supported in part by funds from Indian Council of Medical Research, New Delhi and Grant No. E-3427, National Institute of Allergy and Infectious Diseases U.S.P.H.S. Thanks are due to Dr.R. Viswanathan for his interest in this work.     

Zur Spaltung von Adenosintriphosphat durch die Z-Scheiben der indirekten Flugmuskeln vonPhortnia regina (Diptera)

Summary ATP splitting is demonstrated to occur in the Z discs of isolated flight muscle myofibrils ofPhormia regina after the quantitative extraction of myosin.     

Spermatozoa: models for studying regulatory aspects of energy metabolism

Spermatozoa are highly specialized cells, and they offer advantages for studying several basic aspects of metabolic control such as the role of adenosine triphosphate-(ATP)-homeostasis for cell function, the mechanisms of fatigue and metabolic depression, the metabolic channelling through the cytoplasm and the organization and regulation of glycolytic enzymes. Spermatozoa of four species with different reproductive modes are, introduced and the first results are presented: Spermatozoa of the marine wormArenicola marina are well adapted to external fertilization in sea water with fluctuating oxygen tension: they are motile for several hours in oxygen-free sea water, even when the ATP level is dramatically reduced. Anaerobic ATP production occurs by alanine, acetate and propionate fermentation probably by the same pathways known from somatic cells of this species. Under aerobic conditions the phosphagen system might function like a shuttle for energy-rich phosphate from mitochondria to the dynein-ATPases. Storage of turkey and carp spermatozoa for several hours without exogenous substrates and oxygen results in the degradation of phosphocreatine and ATP to inorganic phosphate and adenosine monophosphate (AMP), respectively. Despite low energy charges, stored spermatozoa of both species are capable of progressive movements. In carp spermatozoa fatigue of motility is not accompanied by the dramatic acidosis one discusses as an important effect in muscle fatigue. Energy metabolism of boar spermatozoa is typically based on glycolysis consuming extracellular carbohydrates and producing lactate and protons. The sperm seem to tolerate low intracellular pH (<6.5). The lack of a phosphagen system (no energy shuttle from mitochondria to the distal dynein-ATPases) is probably compensated by a high glycolytic ATP-production in the mitochondria-free piece of the flagellum.     

Effect of spermine on adenyl cyclase activity of spermatozoa

Zusammenfassung Zusätze physiologischer Sperminkonzentrationen (2–14 mM) zu menschlichen Spermiensuspensionen bewirken eine Steigerung der Adenylcyclase-Aktivität, wie sie durch die vermehrte Bildung von cAMP aus ATP angezeigt wird.     

Photostimulation of ATP production,in cell-free extracts of insect integument

Summary Cell free extracts ofPieris brassicae integument or its 220,000×g sonicated supernatant exhibit a light-dependent ATP production system (LAPS). ADP and Pi seem to be the substrates of the reaction. LAPS cannot be located in intact mitochondria.     

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.     

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC₅₀ = 37.5 μM; K _i = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.     

Myofibrillar ATP-splitting in the elementary contractile cycle of an insect flight muscle

Zusammenfassung In glyzerinierten fibrillären Muskelfasern vonLethocerus werden schon 20 msec nach maximaler «Dehnungsaktivierung» der ATP-ase (um 5000%) 2 Mol ATP pro Mol Myosin gespalten, worauf die Kontraktionsspannung mit der Wechselzahl von Myosin-ATPase und Querbrücken (etwa 10 Hz) oszilliert.     

Analysis of nucleotides bound to the ATP synthetase from spinach chloroplasts isolated under different conditions

Summary The total amount of bound adenine nucleotides in the coupling factor isolated from spinach chloroplasts and its distribution on AMP, ADP and ATP was analyzed after various incubation conditions. During purification of the coupling factor, the distribution of AMP, ADP and ATP is not altered. The coupling factor from deenergized membranes contains approximately 1 ADP, less than 1 ATP, and small amounts of AMP. During phosphorylation the pattern is changed and ATP becomes the dominant species. When exogenous ADP is lacking, phosphate is readily incorporated into ATP. Inhibition of adenylate kinase by AP₅A does not change the distribution pattern of the adenine nucleotides. The distribution pattern shows no integer numbers for the different nucleotides, suggesting that the coupling factor is present in different states in a statistical distribution.Acknowledgment: We thank the Swiss National Foundation for Scientific Research (grant 3.582.79) for generous support.     

Autoradiographic study of the penetration of non-histone chromatin proteins into differentiating cells

Summary Attention has previously been drawn to a specific effect of NHCP on embryonicPleurodeles cell differentiation. With a modified NHCP labelling technique, autoradiography has revealed a cytoplasmic concentration of labelled NHCP and has not revealed any difference between homospecific and heterospecific NHCP penetration.This work has been supported financially by research grants from the C.N.R.S. (ERA No. 327 and ATP No. 4310 on the cellular differentiation), and INSERM (No. 711019).The authors wish to extend their thands to Dr.Mazarguil who kindly initiated us in the techniques of enzyme binding. Many thanks are also due to Mr.Hawks for most valuable help in correcting the English.     

Force generation in glycerinated insect-flight muscles without ATP

Zusammenfassung Durch Zugabe und durch Auswaschen von Mg-Pyrophosphat (0,5 mM) oder Mg-Tripoly-Phosphat (1 mM) konnten (in Abwesenheit von ATP) in glyzerinextrahierten Fasern von fibrillären Insektenmuskeln (Lethocerus maximus) reversible Kontraktionszyklen und Änderungen des Dehnungswiderstandes bewirkt werden.

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Supported by Grant No. Ru 154/b of the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Mrs.Helgard Jung is gratefully acknowledged.