Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Ethylene production and β-cyanoalanine synthase activity in carnation flowers

The relationship between ethylene production and the CN^--assimilating enzyme -cyanoalanine synthase (CAS; EC 4.4.1.9) was examined in the carnation (Dianthus caryophyllus L.) flower. In petals from cut flowers aged naturally or treated with ethylene to accelerate senescence the several hundred-fold increase in ethylene production which occurred during irreversible wilting was accompanied by a one- to twofold increase in CAS activity. The basal parts of the petal, which produced the most ethylene, had the highest CAS activity. Studies of flower parts (styles, ovaries, receptacles, petals) showed that the styles had a high level of CAS together with the ethylene-forming enzyme (EFE) system for converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The close association between CAS and EFE found in styles could also be observed in detached petals after induction by ACC or ethylene. Treatment of the cut flowers with cycloheximide reduced synthesis of CAS and EFE. The data indicate that CAS and ethylene production are associated, and are discussed in relation to the hypothesis that CN^- is formed during the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglyoine - CAS -cyanoalanine synthase - CHI cycloheximide - EFE ethylene-forming enzyme     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Effects of 5'deoxy-5'-methylthioadenosine on the metabolism of S-adenosyl methionine

The treatment of transformed rat cells with micromolar amounts of 5'deoxy 5'methyl thioadenosine induces rapid effects on the rate of methylation of DNA concomitantly with alterations of intracellular pools of S-adenosyl methionine and S-adenosyl homocysteine. Pulse chase labelling experiments indicate that 5'deoxy 5'methylthioadenosine does not inhibit the degradation of S-adenosyl homocysteine but inhibits the consumption of S-adenosyl methionine. In vitro transmethylation assays performed with heterologous DNA show that low doses of the thioethernucleoside do not significantly affect the DNA methyltransferase activity of cellular extracts. The biological role of 5'deoxy 5'methylthioadenosine, a natural molecule formed during the synthesis of polyamines is discussed.     

A new system for ATP-metric immunoanalysis

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252-4261] that the reactivity of the acyl-phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP-insensitive phosphoenzyme.     

Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence of ADP, Mg2+, Pi and PPi the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein-1 X min-1, at 20 degrees C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N'-dicyclohexylcarbodiimide.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.