An improved assay for UDPglucose pyrophosphorylase and other enzymes that have nucleotide products

UDPglucose pyrophosphorylase catalyses the interconversion UDPglucose plus pyrophosphate and glucose 1-phosphate plus UTP. Several assay methods for this enzyme have been described but the only one that can be used to investigate the specificity with respect to various UDPsugars is based on coupling to UTP formation. This assay employs phosphoglycerate kinase to catalyse the formation 1,3-bisphosphoglycerate which is then used to oxidise NADH in the presence of glyceraldehyde 3-phosphate dehydrogenase. We have found that the activity of phosphoglycerate kinase towards UTP is low which limits the usefulness of the assay to very low rates, in agreement with the published recommendation of Hansen et al.⁵. Here it is shown that the dynamic range of the assay is increased by more than five fold on addition of nucleoside diphosphate kinase and ADP, which convert UTP to the preferred phosphoglycerate kinase substrate, ATP. It is also shown that the improved assay is suitable for enzymes with other nucleotide triphosphate products.     

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.     

Habituation to iterative photostimulation in the palmar skin conductance response of mice,its delay by psychoanaleptics

Summary In mice, iterative photostimulation results in habituation, detected in the palmar skin conductance response. Psychoanaleptics delay this habituation in proportion to the dose administered.     

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.     

Inhibition of prodigiosin formation inSerratia marcescens by adenosine triphosphate

Summary ATP, inorganic phosphate and ribose inhibited prodigiosin formation inSerratia marcescens, but adenine did not. ATP was not hydrolyzed by the organism during the experiment.     

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.     

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.     

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.     

Regulatory properties of the citrate synthase fromRhodospirillum rubrum

Summary Citrate synthase, purified 600-fold fromRhodospirillum rubrum, is activated by KCl and inhibited by ATP and NADH; the effect of the latter inhibitor is completely counteracted by AMP and partially counteracted by KCl.This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas de la República Argentina and the Consejo de Investigaciones de la Universidad Nacional de Rosario. JJC, and EM and AIH, are members of the Carrera del Investigador Cientifico of the former and the latter institutions, respectively.     

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.     

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.     

Relationship between the absolute configuration and the biological activity of juvenile hormone III

Summary The activity of the pure 10R (=natural) and 10S enantiomers of juvenile hormone III (JH III) was determined in 3 different bioassays, and the relative binding affinity of the 2 enantiomers to the haemolymph JH-binding protein of the cockroachNauphoeta cinerea was measured. In theGalleria wax test, a local morphogenetic assay, the 10R enantiomer was 5240 times more active than, the 10S enantiomer, 1Galleria unit corresponding to 0.42 pg of 10R-JH III as compared to 2.2 ng for 10S-JH III. In a systemic morphogenetic assay with the cockroachNauphoeta cinerea 380 times less 10R enantiomer was necessary in order to induce detectable juvenilisation (58 ng 10R and 22 g 10S) and in a systemic gonadotropic assay withNauphoeta cinerea 255 times less 10R was needed to induce vitellogenin synthesis in 50% of the insects (6.7 ng 10R and 1710 ng 10S). In the JH-binding protein assay 10R-JH III had an affinity for the JH-binding protein (lipophorin) which was approximately 46 times higher than that of 10S-JH III.     

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     

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.     

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.     

Polypeptide translocation machinery of the yeast endoplasmic reticulum

Proteins enter the secretory pathway by two general routes. In one, the complete polypeptide is made in the cytoplasm and held in an incompletely folded state by chaperoning adenosine triphosphatases (ATPases) such as hsp70. InSaccharomyces cerevisiae, fully synthesized secretory precursors engage the endoplasmic reticulum (ER) membrane by interaction with a set of Sec proteins comprising the polypeptide translocation apparatus (Sec61p, Sec62p, Sec63p, Sec71p, Sec72p). Productive interaction requires displacement of hsp70 from the precursor, a reaction that is facilitated by Ydj1p, a homologue of theEscherichia coli DnaJ protein. Both DnaJ and Ydj1p regulate chaperone activity by stimulating the ATPase activity of their respective hsp70 partners (E. coli DnaK andS. cerevisiae Ssa1p, resepectively). In the ER lumen, another hsp70 chaperone, BiP, binds ATP and interacts with the ER membrane via its contact with a peptide loop of Sec63p. This loop represents yet another DnaJ homologue in that it contains a region of 70 residue similarity to the J box, the most conserved region of the DnaJ family of proteins. In the presence of ATP, under conditions in which BiP can bind to Sec63p, the secretory precursor passes from the cytosol into the lumen through a membrane channel formed by Sec61 p. A second route to the membrane pore that is used by many other secretory precursors, particularly in mammalian cells, requires that the polypeptide engage the ER membrane as the nascent chain emerges from the ribosome. Such cotranslational translocation bypasses the need for certain Sec proteins, instead utilizing an alternate set of cytosolic and membrane factors that allows the nascent chain to be inserted directly into the Sec61p channel.     

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.     

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.