Respiration-driven proton translocation in micrococcus denitrificans

The polarity and stoichiometry of respiration-driven proton translocation was studied by electrometric and spectrophotometric techniques inMicrococcus denitrificans in the context of the energy transduction mechanism in bacterial oxidative phosphorylation.

1. Protons are ejected through the plasma membrane during respiratory pulses and thereafter diffuse slowly back.
2. In presence of ionic species mobile across the membrane (K⁺-valinomycin, K⁺-gramicidin, or SCN^?), limiting→H⁺/O quotients of 8 were obtained with endogenous respiratory substrates, and the rate of translocation (14·3 μg ions of H⁺/sec g cell dry weight) was commensurate with that of respiration optimally stimulated by FCCP at an →H⁺/O quotient of 8.
3. The rate of decay of the proton pulses was greatly increased by FCCP, but there was little or no effect on the →H⁺/O quotient characteristic of the respiratory system.
4. Various interpretations of the observations are discussed, and it is concluded that respiration is probably coupled directly or indirectly to electrogenic proton translocation. The observations are compatible with the chemiosmotic hypothesis of coupling between respiration and phosphorylation.

     

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

     

On the possible role of respiratory activity of Acholeplasma laidlawii cells in sugar transport

1. Out of 20 exogeneous substrates only ethanol and, to a much lesser extent, lactate and pyruvate were shown to be capable of stimulating the respiration of Acholeplasma laidlawii cells. However, none of these substrates changed the initial rate of active transport of 3-O-methyl-d-glucose (3-O-MG).
2. From inhibitory analyses and spectroscopic data, it is apparent that the respiratory chain of A. laidlawii has no cytochromes and is probably not responsible for oxidative phosphorylation.
3. Valinomycin and nigericin stimulated cell respiration only in the presence of K⁺-ions, while monensin stimulated it in the presence of Na⁺-ions.
4. 3-O-MG transport was shown to be sensitive to uncouplers, ATPase inhibitors and arsenate are resistant to a majority of respiratory inhibitors tested. This suggested that there was no relationship between respiration and carbohydrate transport in the A. laidlawii cells. Further evidence was provided by the absence of respiratory stimulation during the transport of non-metabolizing carbohydrates.

     

Proton translation in submitochondrial vesicles

An investigation of proton translocation in submitochondrial vesicles from rat liver has been made under simple experimental conditions. Choline chloride was used both as the oxidizable substrate and the ionic medium for the measurement of activity during oxygen pulse experiments:

1. The passive permeability measured from the decay of proton efflux after an oxygen pulse could be described by a first-order equation. An H⁺/O ratio of 2·5 was obtained for choline oxidation in the presence of oligomycin and/or MgCl₂. Oligomycin decreased the passive proton permeability and respiration, concomitant with an increase in proton uptake. Respiratory control was directly related to the passive proton permeability and inversely related to the magnitude of the proton gradient. The decreased respiration and passive permeability reflecting respiratory control is most evident in the pH rang 5·8–7·5.
2. Preparation of submitochondrial vesicles in the presence of EDTA resulted in proton production during an oxygen pulse given at alkaline pH. Cytochromec enhanced proton uptake by approximately 1 H⁺/cytochromec, but only in the presence of Triton X-100. These results are indicative of the asymmetric behavior of the coupling membrane and provide direct evidence of the participation of electron transport components in proton translocation.

     

The role of mitochondria in modifying the cellular inoc environment: Studies of the kinetic accumulation of calcium by rat liver mitochondria

1. The affinity of ATP-supported Ca²⁺ accumulation for both Ca²⁺ and ATP was determined from initial rate studies employing isolated rat liver mitochondria. TheK _m values for “free” Ca²⁺ and ATP were calculated to be of the order of 2 μM and 100 μM, respectively. TheK _m for ATP decreased as the Ca²⁺ concentration was increased.
2. The curve relating initial rates of Ca²⁺ accumulation to Ca²⁺ concentration was singmoidal in shape; values obtained for the Hill coefficient were in the range 1.5–1.9.
3. Concomitant with the ATP-stimulated accumulation of Ca²⁺, ATP translocation was itselt increased in the presence of Ca²⁺. This stimulation took place independently of Ca²⁺ accumulation.
4. Decreasing the pH of the incubation medium decreased the rate of Ca²⁺ accumulation. This inhibition was competitive in that the affinity of mitochondrial for Ca²⁺ could be altered. The maximal rate of accumulation did not change with change in pH.
5. The permeant anions inorganic phosphate and acetate stimulated the accumulation of Ca²⁺ in a non-competitive manner. Both theV _max and K_m varied when either of the anions were present.
6. The data are discussed in relation to the role that mitochondria play in controlling the cellular ionic environment.

     

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

1. The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source.
2. Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR⁺ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH ₄ ⁺ , and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH ₄ ⁺ . Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl.
3. Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR⁺. The nitrate reductase activity in cells of AD2 and N22DNAR⁺ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR⁺ has identical roles. These roles are suggested to include:
4. The first step in the assimilation of nitrate.
5. Provision of an alternative electron acceptor to oxygen for generating a membrane potential.
6. A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth. This type of nitrate reductase, especially in AD2 and N22DNAR⁺, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli.
7. In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

     

Uptake of guanine by synchronizedChlorella fusca

1. The transport of guanine in autospores of light-dark synchronizedChlorella fusca was studied using radioactive guanine in the concentration range of 4 nM to 50 μM.
2. The transport system was constitutive, it had high specificity for the permeant, and theQ ₁₀ value was in the range of 1.5 to 2.2. At concentrations lower than 0.2 μM the half saturating constant, S_0.5 was 1 μM both for cells kept in dark and cells kept in light. At higher concentrations the S_0.5 of darkened cells was about 0.23 μM, while that of illuminated cells was unchanged. Only above 0.2 μM guanine did illumination of the cells or addition of glucose increase the transport rate.
3. Guanine which had accumulated did not leak out at temperatures below 45°C or by treatment with 10 μM dinitrophenol, which completely inhibited transport. Furthermore, the accumulated guanine did not exchange with exogenous guanine.
4. The guanine accumulated, more than 10⁵-fold over the external concentration, showing that the transport, was active.
5. The initial transport rate per cell revealed annual fluctuations.

     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

Effect of feeding on the respiration rate of the rotifer Brachionus plicatilis

The effect of feeding on the respiration rate of Brachionus plicatilis was studied. Oxygen consumption was determined under two feeding regimes, duplicate feeding and constant feeding. Oxygen consumption rate increased during feeding. The oxygen consumption profile is discussed in relation to the following processes:

1. filtration, mastication, and locomotion during feeding
2. specific dynamic action (SDA)
3. egg formation and routine metabolism.

     

The use of a continuous assay system to show the stimulation of phosphoenolpyruvate production in intact mitochondria by valinomycin and by Mn2+

1. A method for the direct recording of the PEP efflux from isolated mitochondria is described.
2. This method has been used to show the stimulation of PEP efflux by externally added Mn⁺⁺ ions.
3. Valinomycin, uncoupler and oleate were also shown to stimulate PEP efflux.
4. Valinomycin caused an increase in the internal concentration of both PEP and citrate.
5. The results indicate that the major pathway of PEP synthesis in isolated mitochondria is via PEP carboxykinase and the results do not call for an unknown pathway of metabolism.
6. Two interactions between PEP and citrate are described; competition for the mitochondrial interior and the stimulation of PEP production by citrate.

     

Adenine nucleotide pool variations in intact Nitrobacter winogradskyi cells

1. The ATP pool in Nitrobacter winogradskyi cells was determined by means of the luciferin-luciferase enzyme system and the ADP and AMP pools were measured after enzymatic conversion into ATP.
2. In the fist 10 min after addition of nitrite to endogenously respiring cells, which had stood for 5–16 days after completion of the nitrite oxidation, the ATP pool dropped about 60%.
3. During the log phase the ATP pool was approx. 20–40 pmoles/5 μg cell-N. During growth it increased exponentially by 3–4 times the amount until the nitrite had been used up. Subsequently the ATP pool decreased at first rapidly and then more slowly without sinking to 0 in the first 2 months after nitrification.
4. Nitrite oxidizing cells had an energy charge of 0.37 during the log-phase. After approx. 90% of the substrate had been used up the energy charge had reached 0.57.
5. If the CO₂ assimilation was inhibited in growing cultures by increased oxygen partial pressure, nitrite oxidation continued but the ATP pool increased.
6. The ATP pool and the activity of the endogenous respiration decreased by more than 50% during the first hours after the substrate had been used up.

     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Inhibition of carotenoid synthesis in Myxococcus fulvus (Myxobacterales)

1. The inhibitory effects of CPTA, nicotine, DPA, and San 6706 on carotenogenesis in Myxococcus fulvus were investigated.
2. The effects of CPTA, D-nicotine, and L-nicotine were very similar. The action of the drugs wasadditive. The cyclization was inhibited at low doses, the introduction of the hydroxyl group at C-1′ at higher doses. Lycopene accumulated at high drug concentration. The mode of action of the inhibitors is discussed.
3. In a carotenoid mutant of M. fulvus a stimulation of the “7,8-dehydrogenase” by CPTA was observed.
4. The specific carotenoid content of bacteria was increased by DPA due to an enhanced formation of phytoene. At low doses of DPA small amounts of an intermediate carotenoid glucoside ester, a 7,8-dihydro derivative, were detected.
5. DPA was taken up by the plasma membrane. Quantitative removal of DPA by washing was not possible.
6. San 6706 specifically and reversibly blocked the desaturation of phytoene.

     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

Frequency filter properties of lobster chemoreceptor cells determined with high-resolution stimulus measurement

1. We developed a high resolution, on-line stimulus measurement system for accurate control of chemical stimulus applications for Homarus americanus lateral antennule chemoreceptors. Focal stimulus presentations in an electrophysiological preparation with the receptor sensilla intact were measured at small spatial (30 μm) and time (5 ms) scales.
2. We tested 15 receptor cells with ten 100 ms pulses of 10⁴ M hydroxyproline at 0.5, 1, 2 and 4 Hz and with a single 8 s square pulse. Individual cells showed differences in their capabilities to resolve pulses (“flicker fusion”). At 2 Hz stimulation, some cells could follow stimulus pulses while others could not. At 4 Hz, 3 cells could still encode individual stimulus pulses accurately. The population resolved pulses up to 2 Hz; at 4 Hz, the population response to a pulse series approximated the response to a square pulse.
3. Repetitive stimulation caused a gradual decrease in the number of spikes and a gradual increase in first spike latency (“cumulative adaptation”). Increased stimulation frequency resulted in greater cumulative adaptation.
4. Since individual differences in adaptation and disadaptation rates of the receptor cells could not be attributed to measured stimulus variability in situ, lobster chemoreceptor cell populations have intrinsic temporal diversity which, we hypothesize, could be used to analyze pulsatile stimuli that occur in natural turbulent odor plumes.

     

Charakterisierung eines phagenähnlichen Partikels aus Zellen von Nitrobacter

1. Polyhedral particles were isolated from cells of Nitrobacter winogradskyi and of Nitrobacter strains K₁, K₄ and α₁. Their physical and biological properties are characterized.
2. The investigated strains contain polyhedral particles, 1000–1200 Å in size. With increasing age of the culture more particles are found in cells of Nitrobacter. Simultaneously the number of colony producing nitritoxidants decreases.
3. In strain α₁ the loss of the capability to form colonies is connected with partial lysis of the cell and release of particles.
4. A homogeneous fraction of particles was obtained by zone density gradient centrifugation in Tris-Mg-SH-buffer.
5. The polyhedral particles have a sedimentation coefficient of s _w,20 ⁰ =825S and a CsCl-buoyant density of ?²⁵ g/cm³.
6. Based on the determined properties the particles are classified as phage-like Nitrobacter particles Nb₁.

     

Glycolipids and the structure of chlorosomes in green bacteria

1. The cellular content of galactolipids in Chlorobium and Chloroflexus is not related to bacteriochlorophyll content nor to the total amount of chlorosome material in the cells.
2. Chlorosomes of both bacteria were agglutinated by Ricinus lectin and the agglutination was increased after treatment of the chlorosomes with trypsin.
3. When cell free preparations of both bacteria were treated with trypsin prior to centrifugation on sucrose gradients, the resulting chlorosome fractions were less contaminated with material derived from the cytoplasmic membrane than when trypsin was not employed.

     

Growth of Nitrobacter in the presence of organic matter

1. Culture filtrates of heterotrophic bacteria were tested for their stimulatory effect on nitrification of three strains of Nitrobacter.
2. Yeast extract-peptone solution, in which Pseudomonas fluorescens had grown, after removal of the cells was added to autotrophically growing cultures of Nitrobacter agilis; it caused a stimulated nitrite oxidation and growth of Nitrobacter agilis.
3. The degree of stimulation depended on: a) the proportion of the culture filtrate to the autotrophic medium; b) the composition of the complex medium in which Pseudomonas fluorescens had been grown; c) the time the heterotrophic bacterium had been grown in the complex medium.
4. The stimulatory effect was highest with Nitrobacter agilis, less with Nitrobacter winogradskyi and negligible with Nitrobacter K ₄.
5. It was possible to adapt nitrifying cells of Nitrobacter agilis to higher concentrations of yeast extract and peptone. After the nitrite had been completely oxidized the cell-N still increased up to 30% before growth stopped.

     

A study on the rate of water transport of the clam katelysia opima in relation to environmental conditions

1. The neutral red technique was employed to study the rate of filtration in Katelysia opima.
2. The weight specific water filtration was found to be greater for younger clams compared to the older ones.
3. The rate of water filtration increased with decreasing salinity.
4. Water filtration was found to increase as temperature increased, reaching a maximum at 35°C. but then sharply decreasing at 39°C.
5. Light had no significant effect on the rate of filtration.
6. Suspended matter was found to affect the rate of water filtration.
7. The rate of filtration was low at high pH and high in low pH.
8. The rate of water filtration was found to be faster during high tide than during low tide.
9. The presence of the parasitic crab, Pennotheris sp., in the mantle cavity of clams had a marked effect on the particle filtration.
10. Accidental cut of the siphon tips had no effect on the rate of filtration.

     

Dioptrics of the facet lenses in the dorsal rim area of the cricket Gryllus bimaculatus

1. The optics of the corneal facet lenses from the dorsal rim area (DRA) and from the dorso-lateral areas (DA) of the compound eye of the cricket Gryllus bimaculatus were studied.
2. The DRA of the cricket eye contains quite normally shaped facet lenses. The diameter of the facet lens in the DA is 2-fold larger compared to that in the DRA. The radius of curvature of the front surface is distinctly less in the DA facet lenses, as the surface of the facet lenses in the DRA are virtually flat.
3. The averaged axial refractive index of the facet lenses of Gryllus bimaculatus, measured by interference microscopy, was 1.496 ± 0.008 (n = 42) in the DRA and 1.469 ± 0.004 (n = 39) in the DA. The geometrical thickness of the lenses was calculated to be 77 ± 3 μm (n = 42) in the DRA and 56 ± 1 μm (n = 39) in the DA.
4. Analysis of the diffraction pattern obtained with a point light source revealed distinct focusing properties of both the DRA and the DA facet lenses; striking Airy-like diffraction patterns were obtained in both cases.
5. Focal distances measured directly at the backfocal plane were 40 ± 8 μm (n = 84) in the DRA of all the animals studied, and 60–90 μm (n = 62) in DA depending on the animal. Analysis of the diffraction of the point light source yielded very similar focal distances: 40 ± 5 μm (n = 10) in DRA and 81 ± 8 μm (n = 11) in DA. In the DRA, focal distance of the facet lenses was smaller than the cone length, 58 ± 3 μm (n = 9) while in the DA the focal distance matched the effective cone length, 71 ± 5 μm (n = 16).