Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: II. Oxidative phosphorylation

Oxidative phosphorylation was measured in isolated energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus with NADH-mediated electron transport. This dark phosphorylation was similar to photophosphorylation in its sensitivity to uncouplers and energy-transfer inhibitors. However, photophosphorylation was 20- to 50-times more active than oxidative phosphorylation. The $\text{P}\text{O}$ ratio of oxidative phosphorylation was about 0.2. Besides oxidative phosphorylation, adenylate kinase- and ADP-P_i exchange activity were measured in the dark. The ADP-P_i exchange reaction was identified as polynucleotide phosphorylase.     

Effect of 4,6-Dinitro-o-sec-butylphenol on Phosphorus Accumulation and Incorporation in Tomato Leaf Disks

The accumulation and incorporation of externally applied P³² into ATP and the effect of 4,6-dinitro-o-sec-butylphenol (DNBP) on these processes was studied, using tomato (Lycopersicon esculentum Mill.) leaf disks.

P³² was, in most part, actively accumulated into leaf disks with time and was incorporated into ATP and other organic phosphates. DNBP inhibited both P³² accumulation and ATP generation. The amount of inhibition increased with time of incubation.

It is concluded that P³² accumulation is related to ATP generation. Even though DNBP greatly inhibits phosphorus accumulation, there is little or no effect on its retention.

DNBP has the ability to uncouple oxidative phosphorylation. Therefore, it is assumed that its inhibitory effect on phosphate accumulation and generation of high-energy phosphorus esters is related to its inhibition of oxidative phosphorylation.

A method is described which appears to be satisfactory to determine the relative amounts of ATP and ADP in leaf disks labeled with P³².

     

Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

Measurement of the intramitochondrial P/O ratio

Measurements of the initial rate of ATP synthesis and the initial rate of oxygen consumption in mitochondria in which transport of ADP, Pi and ATP were inhibited were used to obtain a value for the intramitochondrial $\text{P}\text{O}$ ratio. With succinate as substrate this method yielded a $\text{P}\text{O}$ ratio of 2.8 for the phosphorylation of intramitochondrial ADP.     

Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium.

A procedure for the isolation of membrane vesicles after sonication of $\text{Halobacterium halobium}$ is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In the light, these vesicles extruded protons. However, on addition of low concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.     

Excitation energy transfer in Anacystis nidulans.

Several natural acyclic sesquiterpenes with capacity for insect growth regulation have been shown to uncouple oxidative phosphorylation in mouse-liver mitochondria. These agents stimulate succinate oxidation, reverse oligomycin-inhibited state 3 respiration, activate ATP-hydrolysis, induce loss of respiratory control and abolish $\text{ADP}\text{O}$ ratio. Permeability of the inner membrane to potassium, sodium, ammonium and chloride ions as well as to protons is also enhanced. Since the structure of these agents precludes protonophoric activity, the possible mechanism of uncoupling by these juvenile hormones is discussed.     

Interaction between the oxidative phosphorylation genes of Escherichia coli k12 and the nitrogen fixation gene cluster of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{unc}$^? mutants uncoupled in oxidative phosphorylation, and thus defective in ATP biosynthesis, and an F′ plasmid carrying nitrogen fixation genes from $\text{Klebsiella pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$⁺_Kp genes in $\text{E. coli}$ K12 does not require coupling of oxidative phosphorylation but needs the contribution of an anaerobic electron transport system involving fumarate reduction. The $\text{nif}$_Kp cluster of genes does not contain functions able to complement a defective Mg²⁺-ATPase aggregate but does contain a function(s) which appears to interact with the $\text{unc}$B^? mutant over the formation of a redox system.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.     

Spectrophotometric determination of fluorophor, protein, and fluorophor/protein ratios in fluorescamine and MDPF fluorescent antibody conjugates.

Goat and rabbit gammaglobulin were labeled with the recently introduced fluorogenic reagents fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (IV), or MDPF, and the molar absorption coefficient at 385 nm of the bound fluorogens was determined indirectly via unbound, hydrolyzed reagent. Based on absorption measurements at 280 and 385 nm, the content of bound fluorogen (F) and protein (P) and the $\text{F}\text{P}$ ratio in dilute solutions of antibody conjugates may conveniently be determined with a nomograph or calculated with individual formulas. The molar $\text{F}\text{P}$ ratio of our preparations was normally between 4 and 14, but it may rise to a maximum of about 20 without loss of antibody specificity. Fluorescence is proportional to absorption (fluorophor concentration).MDPF and fluorescamine offer a number of advantages over conventional fluorophors for antibody labeling, and since the method may be adapted to other proteins, it is to be expected that the method described here will be widely applicable.     

Nigericin-induced stimulation of photophosphorylation in chloroplasts

Amines have been shown recently to stimulate at low concentrations the steady-state rate of photophosphorylation by unbroken chloroplasts (Giersch, C. (1982) Z. Naturforsch. 37c, 242–250). In the present contribution it is demonstrated that not only amines but also the carboxylic ionophores nigericin and monensin at concentrations of 10 and 150 nM, respectively, stimulate the phosphorylation rate. The $\text{ATP}\text{2}\text{e}$ ratio is not decreased upon the addition of nigericin at concentrations that stimulate phosphorylation. Nigericin-induced stimulation is observed only in the presence of sufficient external potassium, indicating that the observed stimulation is unlikely to be a side-effect of the uncoupler but is related to H⁺-K⁺ exchange. The proton permeability of the thylakoid membrane is increased and the proton gradient decreased by amounts of nigericin that stimulate phosphorylation. The membrane potential is not affected in the steady state, indicating that the proton-motive force is slightly reduced upon addition of the ionophore. Data on the proton-motive force were related to maximum values of the phosphorylation potential, which was 45 000–50 000 M^?1 in the absence and 30 000–35 000 M^?1 in the presence of 10 nM nigericin. The observation that the $\text{ATP}\text{2}\text{e}$ ratio is not decreased in the presence of uncoupler-induced proton leakage is suggested to indicate that the thylakoid lumen does not represent a homogeneous phase of constant proton electrochemical potential. The results presented here are in agreement with the chemiosmotic concept as far as energetic aspects are concerned but seem to be at variance with the postulated free mobility of protons inside the thylakoids. A tentative model of uncoupler-induced stimulation of phosphorylation is presented.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

Phthalate ester hydrolases and phthalate ester toxicity in synchronously developing larvae of the brine shrimp (Artemia)

Di-n-butyl phthalate (DNBP) is toxic to synchronously developing larvae of the brine shrimp, $\text{Artemia}$. The LD50 for 24 h exposure is approximately 30 μM (8 ppm). DNBP is concentrated by larvae, and maximal uptake of DNBP precedes the onset of mortality. At the time of maximal uptake, most of the DNBP remains in the form of the diester. By the time of maximal mortality nearly all of the DNBP has been converted to the monoester, n-butanol and possibly other polar metabolities. n-Butanol and mono-n-butyl phthalate directly incubated with larvae were nontoxic when tested in concentrations at which DNBP was toxic. Soluble enzyme(s) extracted from the hatched larvae, but not from the dormant embryos, can convert DNBP to its monoester and n-butanol. The amount of enzyme activity increased with larval development in parallel with the kinetics of acute toxicity. The enzyme may be significant in the developmental program as well as in the mediation or moderation of the toxic effects of DNBP in $\text{Artemia}$ larvae.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C., reinhardtii

An anaerobic incubation period of varying duration is required to induce hydrogenase activity in $\text{C.}\text{,}\text{reinhardtii}$. Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.     

Active sodium extrusion reduces net efficiencies of oxidative phosphorylation in the strictly photoautotrophic cyanobacterium Anacystis nidulans

Efficiencies of oxidative phosphorylation ($\text{P}\text{O}$ ratios), intracellular high-energy phosphate pools (ATP and ADP) under aerobic and anaerobic dark conditions, and photosynthetic oxygen evolution measured with intact cells of Anacystis nidulans were found to be specifically depressed by NaCl, but not by KCl. A scheme is proposed which explains the deleterious effect of sodium on the energy metabolism of A. nidulans by competition for protons between ATP synthesis and active sodium extrusion.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.