Quantitative analysis of uncoupling activity of substituted phenols with a physicochemical substituent and molecular parameters

The uncoupling potency of a series of substituted phenols with rat-liver mitochondria was analyzed quantitatively with physicochemical substituent and molecular parameters such as log P, P being the partition coefficient in a phosphatidylcholine liposome/water system, log KA, KA being the acid dissociation constant, and the Taft-Kutter-Hansch steric constant, Es, for ortho-substituents. The potency evaluated from the concentration in the medium required for a defined response was analyzed, showing that the incorporation of compounds in terms of log P, a certain balance between neutral and ionized forms expressible by a parabolic function of log KA and the steric shielding effect of the ortho-substituents on the negatively charged center of ionized form are highly significant factors governing the variations in potency. The potency was also quantitatively separated into the intrinsic potency as the protonophore inside the inner mitochondrial membrane and the incorporation factor in terms of log P. Some phenols found as outliers from the correlations and some others distorting the quality of the correlations were shown to have inhibitory effects on the respiratory chain by specific and non-specific modes of action, respectively, besides uncoupling activity.     

Quantitative relationship between protonophoric and uncoupling activities of substituted phenols

The protonophoric activity through liposomal membranes was measured and compared with the uncoupling activity with the oxidative phosphorylation of rat-liver mitochondria for 19 substituted phenols. Quantitative analyses of the protonophoric activity of the phenols in terms of physicochemical molecular parameters showed that the activity was mostly decided by two factors: the partition coefficient between the liposome and aqueous buffer phases and the acid dissociation constant. Correlation was excellent between protonophoric and uncoupling activities when the difference in the effect of acidity of phenols between liposomal and mitochondrial membranes was taken into account. The results were further evidence for the shuttle-type of mechanism of weakly acidic uncouplers based on the Mitchell chemiosmotic hypothesis.     

The uncoupling activity of substituted anthracene derivatives on isolated mitochondria from Phaseolus aureus

Unsubstituted anthraquinones and anthrones have a low uncoupling activity on isolated mitochondria. The presence of phenolic groups is a prerequisite for a marked effect. The number of phenolic groups is of minor importance but their substitution pattern is important. The methylene group of the anthrones increases the activity.     

Structural requirements of salicylanilides for uncoupling activity in mitochondria: Quantitative analysis of structure-uncoupling relationships

The uncoupling activities of more than 20 salicylanilides were measured in rat liver mitochondria. The activities, expressed as the minimum concentrations required for full release of state-4 respiration, ranged over three orders of magnitude. The acid dissociation constant, pK_A, and the partition coefficient between octanol and water (P_oct) of some of the salicylanilides were determined. These two parameters were found to be well expressed in terms of the Hammett constant, σ, and the hydrophobic substituent coefficient, Π, respectively. The pK_A and log P_oct values of all the salicylanilides were predicted according to these relationships. Furthermore, the capacity factor, k′, on high-performance liquid chromatography was determined on glyceryl-coated-controlled pore glass (gly-CPG). Values of log k′ correlated well with those of log P_oct. The uncoupling activities of the salicylanilides were analyzed in terms of these three parameters. Both hydrophobic and electron-withdrawing properties were found to be essential for induction of potent uncoupling activity. The correlations using log k′ were better than those using log P_oct.     

Quantitative aspects of proline utilization during flight in tsetse flies

ABSTRACT. The proline concentration of the haemolymph in resting tsetse flies provides a reasonable indication of their total proline content. Estimates of pre-flight proline content obtained on this basis were compared with the proline content of flies that had been flown for different durations to provide an estimate of the rate of proline consumption at different stages of flight. The results indicate that the apparent ability of tsetse flies to continue flight after their proline reserves have been exhausted is an artefact of experimental procedure. It is concluded that the flight capacity of tsetse flies is effectively limited by the magnitude of their proline reserve, although this reserve is capable of being supplemented to some extent by the limited synthetic capacity of the fat body.     

Activities of hormone metabolizing enzymes in house flies treated with some substituted urea growth regulators.

The insect growth regulators (IGR) TH 6038 and TH 6040 affect larvae of various species by interfering with cuticle development. In a biochemical study of their effects, larvae of the house fly, $\text{Musca domestica}$ L. were reared for 2 days on diets containing 1.7 to 166.7 ppm of these compounds, then assayed for activities of the microsomal oxidases and the enzyme(s) which metabolize β-ecdysone. The activities of these enzymes were compared with the percentage of treated larvae completing pupal-adult ecdysis. The two compounds reduced the activity of the β-ecdysone metabolizing enzyme(s) by as much as 57%, reduced pupal-adult ecdysis by 43% to 100%, and stimulated microsomal oxidase activity 4- to 12-fold. Supplementation of the diet of the treated insects with the Cecropia juvenile hormone, JH I, partially restored pupal-adult ecdysis but supplementation with β-ecdysone had no effect. The mode of action indicated by these results is that the IGRs cause an accumulation of β-ecdysone in the treated larvae. This stimulates the enzyme, chitinase, which degrades chitin in preparation for formation of the new cuticle. The hormone may also cause a JH deficiency and the stimulation of DOPA decarboxylase and phenol oxidase which would further disrupt the normal molting process.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

Anion permeation limits the uncoupling activity of fatty acids in mitochondria.

The action of such membrane-permeant cations as tetraphenyl phosphonium and dibenzyldimethyl ammonium upon fatty acid-uncoupled respiration has been studied with oligomycin-inhibited rat liver mitochondria. Both cations enhance fatty acid-stimulated respiration. This synergistic effect is explained by a facilitated permeation of the fatty acid anion across the inner membrane due to an ion-pair complex. It is concluded that fatty acid uncoupling in rat liver mitochondria is limited by fatty acid anion permeation.     

Cyclosporin a inhibits the protonophoric uncoupling activity of laurate in liver mitochondria

The effects of cyclosporin A, carboxyatractylate, and glutamate on the protonophoric uncoupling activity of laurate in liver mitochondria have been studied. It was found that 5 μM cyclosporin A partly inhibits laurate-stimulated mitochondrial respiration, which is suggestive of its recoupling effect, i.e., the ability to suppress the protonophoric activity of this fatty acid. Under these conditions, cyclosporin A has no effect on the ability of carboxyatractylate and glutamate to inhibit the uncoupling effect of laurate. In their turn, these compounds do not influence the recoupling activity of cyclosporin A. The recoupling effects of cyclosporin A, carboxyatractylate, and glutamate are additive: acting simultaneously, they fully suppress the uncoupling activity of laurate. It is concluded that the protonophoric uncoupling activity of fatty acids in liver mitochondria is mediated not only by ADP/ATP and aspartate/glutamate antiporters, but also by a system that is sensitive to cyclosporin A, but is not related with cyclophilin D.     

Olfactory responses of tsetse flies to phenols from buffalo urine

Abstract A comparison was made of the EAG responses of males and females of Glossina morsitans morsitans Westwood, G.austeni Newstead and G.tachinoides , Westwood to various doses of compounds known to be components of ox and buffalo urine fractions which are attractive to tsetse in the field (phenol, 3- and 4-methylphenol, 3- and 4-ethylphenol, 4-n-propylphenol, dimethylsulfone). All three species did not respond to dimethylsulfone. The overall responses to the phenolic substances were higher in females than in males in G.m.morsitans and higher in males than in females in G.austeni and G.tachinoides. Response spectra of the species for the phenolic substances suggested that G.m.morsitans and G. austeni were most responsive to 3- and 4-methylphenol and 3-ethylphenol, whereas G. tachinoides was most sensitive to 3-ethylphenol and 3-methylphenol, and only moderately sensitive to 4-methylphenol.
Cross-adaptation experiments, in which l-octen-3-ol, acetone, 4-heptanone and 3-nonanone were also included, revealed that all phenolic compounds stimulated one and the same class of receptors, which differed from the class of receptors activated by l-octen-3-ol. The ketones also had their own receptors. Hence, the flies can obtain information about the presence of attractants by at least three different receptor classes. It was concluded that phenol and any individual alkylphenol found in ox and buffalo urine should be attractive to tsetse flies, provided that stimulus intensity is above threshold and not beyond optimum. One class of receptors may respond more strongly in males than in females, whereas another class is more responsive in females than in males. This may result in a change in sex ratios in catches depending on the odour bait used.     

Bioenergetics of mitochondria from flight muscles of aging blowflies: partial reactions of oxidation and phosphorylation.

Mitochondria from flight muscles of senescent blowflies, Phormia regina, which exhibit decreased rates of coupled, or ADP-stimulated, oxidation of α-glycerolphosphate or pyruvate show similar decreased rates of uncoupled, or carbonylcyanide-p-trifluoromethoxyphenylhydrazone-stimulated oxidation. Thus, uncoupled α-glycerolphosphate oxidation is decreased by 20% and uncoupled pyruvate oxidation by 39% in mitochondria isolated from 31- to 33-day-old blowflies as compared with mitochondria from 7- to 9-day-old flies. The finding of nearly equal decreases with age in coupled and uncoupled respiration suggests that the age-dependent defect lies within the oxidative or electron transport pathway, and not associated with phosphorylation. However, no such change with age is observed in any of the partial reactions of electron transport that were examined. These include the following partial reactions: (a) pyruvic dehydrogenase; (b) pyruvate-ferricyanide reductase: (c) α-glycerolphosphate dehydrogenase; (d) α-glycerolphosphate-ferricyanide reductase; and (e) cytochrome oxidase. In addition, no age-associated decline is observed in the content of cytochromes b, c + c₁, a, or a₃ or in the specific activity of the fully activated mitochondrial ATPase (assayed in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone). However, the specific activity of the masked ATPase (assayed in the absence of uncoupler) is increased 38% in mitochondria from senescent blowflies.     

Unique features of flight muscles mitochondria of honey bees (Apis mellifera L.)

Honey bees Apis mellifera L. are one of the most studied insect species due to their economic importance. The interest in studying honey bees chiefly stems from the recent rapid decrease in their world population, which has become a problem of food security. Nevertheless, there are no systemic studies on the properties of the mitochondria of honey bee flight muscles. We conducted a research of the mitochondria of the flight muscles of A. mellifera L. The influence of various organic substrates on mitochondrial respiration in the presence or absence of adenosine diphosphate (ADP) was investigated. We demonstrated that pyruvate is the optimal substrate for the coupled respiration. A combination of pyruvate and glutamate is required for the maximal respiration rate. We also show that succinate oxidation does not support the oxidative phosphorylation and the generation of membrane potential. We also studied the production of reactive oxygen species by isolated mitochondria. The greatest production of H₂O₂ (as a percentage of the rate of oxygen consumed) in the absence of ADP was observed during the respiration supported by α‐glycerophosphate, malate, and a combination of malate with another NAD‐linked substrate. We showed that honey bee flight muscle mitochondria are unable to uptake Ca²⁺‐ions. We also show that bee mitochondria are able to oxidize the respiration substrates effectively at the temperature of 50°С compared to Bombus terrestris mitochondria, which were more adapted to lower temperatures.