The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

Maintenance of energy-linked functions in rat liver mitochondria

EGTA and EDTA are compared with respect to their ability to preserve ATP-requiring reactions in rat liver mitochondria. The presence of EGTA sustains the high ATP requirement of citrulline synthesis. EDTA does not, even with excess Mg2+. Carboxylation of pyruvate, which has a lower ATP demand, is not influenced by the type of chelating agent. In mitochondria stored for 24 h at 4 degrees C, EGTA is more effective than EDTA in preventing loss of these energy-linked functions.     

The effect of equisetin on energy-linked reactions in Rhodospirillum rubrum chromatophores

Light-induced proton uptake, light-induced carotenoid absorbance shift, photophosphorylation, and hydrolysis of Mg-ATP, Ca-ATP, and PPi in Rhodospirillum rubrum chromatophores are shown to be inhibited by the antibiotic equisetin. The Mg- and Ca-ATPase activities of purified F0F1-ATPase are inhibited by equisetin. In contrast, only the Ca-ATPase activity of purified F1-ATPase is decreased by equisetin, whereas the Mg-ATPase is stimulated. Both equisetin and N,N'-dicyclohexylcarbodiimide (DCCD) inhibit the hydrolytic activity of the purified H+-PPase but not the hydrolytic activity of soluble PPase from R. rubrum and yeast. The I50 for the PPi hydrolysis is near 20 microM for both equisetin and DCCD. The action of equisetin on membranes is compared to the effect of Triton X-100 and carbonyl cyanide p-trifluoromethoxyhydrazone. On the basis of these new data, equisetin is proposed to act nonspecifically on membranes and hydrophobic domains of proteins.     

Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.     

Restoration of energy-linked functions in "aging" rat-liver mitochondria

Respiratory control, lost by rat-liver mitochondria during storage at 0°, can be restored if the mitochondria are incubated under energy-generating conditions for 2 hr at 25°C. The extent of restoration is virtually complete in 1 day old mitochondria but diminishes with aging thereafter. Under the conditions employed nupercaine had no effect on either the aging or the restorative functions.     

Membrane mutation affecting energy-linked functions inEscherichia coli K 12

A small-colony forming variant ofEscherichia coli with a mutation in thenof gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicina indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg²⁺-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.     

The effect of dimethyl sulphoxide on electron transport in chloroplasts

The effect of DMSO (dimethyl sulphoxide) on electron transport in chloroplast membranes has been studied. It has been found that concentrations of DMSO up to 20% (v/v) do not inhibit electron transport in freshly isolated chloroplasts, but that higher concentrations start to cause inhibition. However, in chloroplasts that have been aged for 8 to 24 hours by storage at 4 degrees C, the addition of DMSO at concentrations up to 20% causes stimulation of electron transport. Possible mechanisms for this effect are discussed.     

Alteration of energy-linked functions in rat hepatic mitochondria following aflatoxin B1 administration

Respiratory activity in hepatic mitochondria have been examined following administration of the carcinogen aflatoxin, (AFB₁) to rats. Measurement in isolated mitochondria of respiration rates in presence of ADP (state 3) and after its depletion (state 4) revealed that these rates were not significantly altered in livers of rats obtained 4–8 hours after single injection of AFB₁ (7 mg/kg of body weight). After 12–24 hours, however, a generalized inhibition in state 3 respiration rate and ADP phosphorylation rate had been evident with several FAD- and NAD-linked oxidizing substrates. But the ADP:0 ratio did not show any alteration. State 4 respiration rates, on the other hand, were increased remarkably (38–94% depending on substrate used), thereby recording in each case a decrease in respiratory control ratio (state 3:state 4 ratio), indicating probable damage to mitochondrial membrane as a result of AFB₁ ingestion. This was also evident from greater basal ATPase and Mg²⁺-ATPase activities and low total ATPase activity. After 48–72 hours of AFB₁ treatment, the respiratory rates as well as the ATPase activities returned to normal levels, suggesting probable recovery of mitochondrial functions from the toxic effects of AFB₁. © 1997 John Wiley & Sons, Inc.     

Preservation of energy-linked functions of insect mitochondria by simple freezing methods.

Attempts are made to preserve some energy-linked functions of mitochondria isolated from the fat body of the cockroach Nauphoeta cinerea. The ability of mitochondria to sustain appreciable levels of oxidation, phosphorylation, respiratory control and 2, 4-DNP-stimulation of respiration which are normally lost during incubation at 30°C for 2 hrs or during aging at 0–2°C for 48 hrs can be preserved effectively by storing mitochondrial suspensions in sucrose - EDTA medium for 1 week at - 20°C and at least 3 weeks at -196°C (liquid nitrogen temperature). DMSO (dimethyl sulfoxide; 10% v/v) was found to be ineffective as an aid to preservation at 0–2°C, a significant aid at -20°C and unnecessary at -196°C. With slight variations (for flight muscle mitochondria which requires DMSO at -196°C), this procedure is also effective for mitochondria isolated from other tissues of various insect species. EDTA was found to be an essential ingredient of the isolation medium and therefore for all storage procedures. Both lyophilization and the preincubation of mitochondria with nupercaine (400 μM) have deleterious effects.     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.