Addition Of DNP Research Articles

Evaluation of the Integrity of the Respiratory Chain in Rooster Sperm after Freezing

One of the causes of cell death is a violation of electron transfer through the respiratory chain of mitochondria. The aim of study was to assess the integrity of the respiratory chain sperm after freezing in roosters. The effect of 2.4 dinitrophenol (2.4 DNP) on NADH fluorescence in fresh and cryopreserved semen of roosters was investigated. If the transfer of electrons and protons along the respiratory chain functions normally 2.4 DNP enhances the nonphosphorylating oxidation of NADH and its fluorescence decreases. The sperm of 30 cocks was frozen. The NADH was measured on a LUMAM fluorescence microscope. The luminescence excitation wavelength of NADH is 365 nm. Registration of NADH was carried out at a wavelength 465 nm. The amount of NADH was expressed in conventional units and the change in fluorescence after addition of 2.4 DNP was calculated as a percentage of the initial value taken as 100 %. The fluorescence of NADH after adding 2.4 DNP to frozen semen was 9% less than in fresh semen. A relationship between the magnitude of the change in NADH fluorescence after the addition of DNP and egg fertilization was established (r =0.65; P<0.05). Respiratory stimulation by 2.4 DNP is an useful and informative indicator in assessing sperm quality.Support or Funding InformationThe study was supported by the Russian Science Foundation (project No. 18‐16‐00071)

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Activation of the PHD oxygen‐sensing pathway induces a low‐respiring, protected mitochondrial phenotype

In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the proline hydroxylase-domain containing (PHD) oxygen-sensing pathway in cardiomyocytes. Addition of cyanide and the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused, as expected, the collapse of mitochondrial membrane potential (ΔΨmito) in control cells with only partial recovery upon washout. In contrast, ΔΨmito is partially maintained during metabolic inhibition and recovers completely upon washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse ΔΨmito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of ΔΨmito in PHI-treated myocytes, while a role for the ‘reverse mode’ operation of the F1Fo-ATP synthase was ruled out. Evidence of fumarate to succinate reduction by complex II in the PHI-treated, but not control myocytes, provides an explanation for the apparent finding of ‘anaerobic’ complex I function. Finally we find that PHI-treatment downregulates basal O2-consumption to only ~15% that of controls. Remarkably, addition of DNP, an uncoupler, does not increase O2-consumption rates in PHI-treated myocytes. We conclude that PHD pathway directs mechanisms that allow anaerobic complex I function and that lead to a low-respiring mitochondrial phenotype that is protected against metabolic insult.

Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts

In cattle embryos, development to the blastocyst stage is improved in the presence of 10 micro;m 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1alpha and 2alpha (HIF1alpha, HIF2alpha) mRNA. Although HIF1alpha protein remained undetectable in bovine blastocysts, HIF2alpha protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 microm DNP improves the quality of bovine embryos, coincident with increased HIF2alpha protein localisation within ICM cells and increased HIFalpha mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

Stereoselective transport of histidine in rat lung microvascular endothelial cells

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.

Both dinitrophenol and Ba 2+ reduce KCl and fluid secretion in Malpighian tubules of Formica polyctena: The role of the apical H + and K + concentration gradient

In the present study, further evidence is presented for the close relationship between fluid secretion and the ratio of the apical H + over K + concentration gradient. Two agents with a fast and reversible inhibitory effect on fluid secretion were tested on intracellular and luminal H + and K + concentrations and on transepithelial and transmembrane potential differences: 6 mM Ba 2+, a K + channel blocker, and 2·10 −4 M, 2,4-dinitrophenol (DNP), a well-known protonophore and uncoupler of oxidative phosphorylation. Ba 2+ hyperpolarized the apical membrane potential difference (V ap, lumen reference) from −59 ± 3 to −90 ± 6 mV ( n = 6) and the basal membrane potential difference (V bl) from −23 ± 3 to −74 ± 7 mV ( n = 6). At the same time, the cell acidified (H c increased from 18 ± 4 to 50 ± 21 nM and the lumen alkalinized (H l decreased from 117 ± 23 to 74 ± 11 nM); H l/H c was reduced from 7.7 ± 2.4 to 2.8 ± 0.8 ( n = 6). On the other hand, K c dropped from 85 ± 6 to 73 ± 6 mM ( n = 9) and K l from 143 ± 3 to 121 ± 1 mM ( n = 5); consequently K l/K c remained unchanged (i.e. 1.7 ± 0.1). As a result, the H l/H c over K l/K c ratio decreased from 4.5 to 1.7. DNP depolarized V ap from −63 ± 7 to −26 ± 3 mV ( n = 8); V bl slightly depolarized from −21 ± 1 to 20 ± 1 mV. In the presence of 6 mM Ba 2+, V ap and the basal membrane potential difference (V bl) (bath reference) depolarized from −81 ± 5 to 1 ± 2 mV and from −68 ± 6 to 1 ± 1 mV, respectively ( n = 5) when applying DNP. Like Ba 2+, the addition of 2·10 −4 M DNP to the control solution caused an acidification of the cytosol (H c rose from 24 ± 5 to 81 ± 9 nM); H l was not significantly changed (i.e. 80 ± 12 and 80 ± 9 nM in the absence and presence of DNP, respectively). Consequently, H l/H c dropped from 3.0 ± 0.7 to 1.0 ± 0.2 ( n = 8). K c diminished from 104 ± 9 to 80 ± 5 mM and K l from 141 ± 8 to 93 ± 6 mM after the addition of DNP; K l/K c was not significantly changed (i.e. 1.4 ± 0.1 and 1.2 ± 0.2 in the absence and presence of DNP, respectively, n = 6). The overall result was a reduction of the ratio (H l/H c over K l/K c) from 2.1 to 0.8. On the other hand, the sensitivity of V bl and V ap to a change in bath K + from 5 to 51 mM was virtually unchanged from control in the presence of 2·10 −4 MDNP: V bl depolarized with 33 ± 1 and 32 ± 1 mV, and V ap with 19 ± 3 and 22 ± 1 mV, in the absence and presence of DNP, respectively. Furthermore, the transient changes of V bl on varying the bath K +, suggesting a change in K c, were comparable whether DNP was present or absent. These findings are consistent with the hypothesis of an apical K +/H + antiporter. Ba 2+ and DNP reduce the driving force for K + secretion to the lumen by slowing down the K +/H + antiporter. Ba 2+ increases the electrical component of the proton motive force of the electrogenic H + pump, thereby decreasing the free energy for building up a proton concentration gradient. DNP inhibits the realization of an apical lumen to cell directed H + concentration gradient by a double action mechanism. It depletes the apical H + pump from its energy source, and it can dissipate H + gradients via its protonophore action at the basal (and apical) cell membrane(s). Further evidence is presented for the electroneutrality of the K +/H + antiporter.

Salt stress and respiration inAspergillus repens

Studies with whole cells and mitochondrial fractions revealed increased respiratory activity inAspergillus repens grown under salt stress conditions. The state 3 and state 4 respiration rates, P∶O ratios, and Mg2+-dependent ATPase were higher in mitochondria of stressed cells than in control cells.A. repens respires via an antimycin A-and cyanide-sensitive pathway. Oligomycin, dicyclohexylcarbodiimide (DCCD) and rotenone inhibited respiration rates less in mitochondria of stressed cells than in controls. Though 2,4-dinitrophenol (DNP), carbonyl cyanide-m-chlorophenylhydrazone (m-Cl-CCP), and carbonyl cyanide-p-trifluoromethylhydrazone (p-F3-CCP) did not stimulate Mg2+-ATPase activity, DNP enhanced the respiration rates, whereasm-Cl-CCP andp-F3-CCP decreased the respiration rates in either condition; mitochondria of stressed cells exhibited a lower degree of inhibition than controls. Addition of DNP, oligomycin, and DCCD inhibited the basal Mg2+-ATPase (ATPase activity without Mg2+ addition). Oligomycin inhibited the Mg2+-ATPase. DCCD showed less inhibition in mitochondria under stress than did the controls. Levels of some respiratory enzymes were higher in the culture grown under stress than in the controls.

The effect of a metabolic inhibitor upon the properties of the cerebral vasculature during a whole-head saline perfusion of the rat.

The effect of the metabolic inhibitor 2,4-dinitrophenol (DNP) has been assessed during a simple in situ Ringer solution perfusion of the rat brain. The preparation was perfused, with or without the addition of DNP, for periods ranging up to 30 min. Following this pre-test perfusion, both the vascular permeability and cerebral perfusate flow were assessed. In the absence of DNP significant barrier disruption had taken place by 10 min and the flow rates showed greater fluctuations with time. In the presence of DNP, however, perfusate flow remained constant and the blood-brain barrier remained intact to [14C]mannitol for at least 10 min, but subsequently the flow rate dropped and the barrier began to show evidence of disruption. The unbound visual marker, Evans Blue, was apparently excluded from all regions other than those that are known to lack a blood-brain barrier. The water content of the brain showed no significant increase until 20 min. Patency of the capillaries was demonstrated by direct visualization of the cerebral vasculature with an Indian ink-gelatin mixture and in some animals there was evidence of incomplete filling following 30 min of perfusion. It is concluded that the use of DNP in the perfusate provides a useful preparation for the short-term study of passive properties of the blood-brain barrier, such as carrier-facilitated diffusion, as well as mechanisms of barrier opening.

Effect of dinitrophenol and anoxia on isometric tension in rabbit colon smooth muscle.

In a previous study, it was suggested that inhibition of oxidative phosphorylation in smooth muscle is accompanied by a release of Ca2+ from mitochondria (Pettersson 1983); in this investigation, the effects of 2,4-dinitrophenol (DNP) and anoxia on isometric tension in pieces of smooth muscle from rabbit colon were studied. Addition of DNP to the bathing medium elicited a transient and dose-dependent contraction with an ED50 value of 2 X 10(-4) M. The contraction was not inhibited by pretreatment with atropine (1 X 10(-6) M). The removal of external Ca2+ did not reduce the contracting action of DNP. Addition of the local anaesthetic D-mepivacaine (1 X 10(-3) M), which reduces the Ca2+ efflux through the plasma membrane of the smooth muscle cells, augmented the response to DNP. Gassing the bathing medium with 95% N2 + 5% CO2 (anoxia) for 60 min. did not affect the basal tension, but markedly reduced the contractile response to DNP. The results indicate that DNP increased cytoplasmic Ca2+ significantly enough to induce a contraction of rabbit colon smooth muscle by a large release from an intracellular store, probably from the mitochondria. Since anoxia did not mimic the contracting action of DNP, it is suggested that a high concentration of DNP would be a more effective stimulus for mitochondrial Ca2+ release than anoxia.

Effects of Dinitrophenol on Phosphorylase a Activity, Adenine Nucleotide Levels and Tension in Rabbit Colon Smooth Muscle

Glycogen phosphorylase a activity, the contents of adenine nucleotides and isometric tension were measured in rabbit colon smooth muscle after exposure to 2,4-dinitrophenol (DNP). DNP caused a dose-dependent increase in phosphorylase a activity, with an ED50 value of 1 X 10(-4)M. Since adenine nucleotides, especially 5-AMP by inhibiting the phosphorylase a to b conversion, might increase the phosphorylase a activity, a time-response study was undertaken in order to analyse the time course of changes in the adenine nucleotide content and phosphorylase a activity after addition of DNP. The latter activity was increased after only 1 min. of incubation. At this time no changes were found in the contents of ATP, ADP, 5-AMP or CP. Not until the phosphorylase a activity had reached its maximum after 5-7.5 min. did the 5-AMP content increase. The phosphorylase a activity then started to decline, but the 5-AMP content continued to rise. The effect of DNP on isometric tension was also studied to test whether the cytoplasmic Ca2+ concentration was increased. An increase in tension was observed 5 min. after administration of the drug and was maximal at 15 min. The results seem to dispute against 5-AMP as being a mediator for the DNP-stimulated increased in phosphorylase a activity in smooth muscle. The finding that DNP elicited a contraction of rabbit colon supports the earlier suggestion that the cytoplasmic Ca2+ concentration was increased (Pettersson 1983).

Metabolic factors regulating the generation of prostaglandins E 1 and E 2 by isolated rat uterus

The generation and output of PGE 2 and PGE 1 by isolated rat uterus incubated with or without glucose as well as the influence of different metabolic substrates and selective enzyme inhibitors on tissue formation and release of PGE material of the 1 and 2 series, were explored. The output of PGE 2 was comparable with glucose, fructose, lactate or pyruvate as the substrate but diminished significantly in citrate-containing solution. In substrate-free media uteri released more PGE 2 than in the presence of glucose or other substrates. The PGE 1 Liberated was similar in glucose, fructose, lactate or pyruvate and decreased significantly in citrate or without substrate. 2-deoxy-D-glucose (2-DG), fluoride or arsenite failed to alter significantly the release of PGE 2 whereas 2,4-dinitrophenol (DNP) evoked augmentation. The uterus released less PGE 1 after 2-DG or fluoride but not following the addition of DNP or arsenite. The results suggest that the synthesis and release of PGE 2 and PGE 1 in the isolated rat uterus appear to be selectively modulated by different tissue metabolic pathways.

Formation of branched chain acylcarnitines in mitochondria

1. 1. The formation of acylcarnitines in isolated mitochondria incubated with L-[ Me- 3H]carnitine and the branched chain 2-oxoacids corresponding to valine, leucine and isoleucine has been studied. 2. 2. Hitherto unknown acylcarnitines were formed and identified as isobutyrylcarnitine (from 2-oxoisovalerate), isovalerylcarnitine (from 2-oxoisocaproate) and 2-methylbutyrylcarnitine (from 2-oxo-3-methylvalerate). 3. 3. The addition of DNP or ADP + P i to the incubation medium increased the formation of propionylcarnitine from 2-oxoisovalerate. 4. 4. The carnitine acetyltransferase (EC 2.3.1.7) was found to transfer acyl groups from branched chain acylcarnitines, but the transfer rates were lower than those found when the corresponding straight chain acylcarnitines were the substrates. 5. 5. Liver, kidney and heart mitochondria from the rat and liver mitochondria from the mouse were all found to have branched chain 2-oxoacid dehydrogenase activity.

The effect of tannic acid on the phosphorylation and ATPase activity of mitochondria from blowfly flight muscle

Low concentrations (below 10 −6M) of tannic acid reduced the respiratory control index of blowfly flight muscle mitochondria, apparently reducing the rate of phosphorylation, although ADP:O ratios were normal. At higher concentrations, no stimulation in respiration was found when ADP was added. Respiration uncoupled with DNP was only slightly affected by tannic acid, even at high concentration. However, when DNP is added after tannic acid, its effect is dependent both on the concentration of tannic acid and on the time that elapses before the addition of DNP. Tannic acid also inhibited the mitochondrial ATPase activity, the DNP-stimulated activity being particularly sensitive to low concentrations. Comparisons are made with the action of oligomycin and the results are discussed in relation to the effect of tannic acid on erythrocytes.

The influence of 2,4-dinitrophenol on metabolic changes caused by ethanol in the perfused rat liver.

The experiments were undertaken in order to investigate the mechanism by which ethanol inhibits the tricarboxylic acid cycle. Isolated livers were perfused with human erythrocytes suspended in an artificial “plasma”. Ethanol was added 45 min. after the start of the perfusions. In some experiments dinitrophenol was added after ethanol. The O2 consumption in the perfused rat liver was unaltered by the addition of ethanol to the medium, whereas the CO2 production fell considerably, resulting in a decrease in the RQ from 0.8 to 0.4. The addition of ethanol caused an increase in both lactate/pyruvate and hydroxybutyrate/acetoacetate ratios in the perfusion medium. When DNP was added to the perfusion medium, half an hour after the addition of ethanol, both the O2 uptake and CO2 production increased. The RQ increased from 0.4 to 0.6. The lactate/pyruvate ratio was increased by the addition of DNP because the amount of lactate liberated from the liver was increased, while the pyruvate concentration was almost unchanged. The addition of DNP caused a fall in the hydroxybutyrate/acetoacetate ratio from 2.2 to 0.6, and a net disappearance of ketone bodies was observed. An opposite change in the extra‐ and intramitochondrial NADH/NAD ratio was observed, favouring the hypothesis that they are really independent of each other. A correlation between the intramitochondrial NADH/NAD ratio and the tricarboxylic acid cycle inhibition was demonstrated.

EFFECTS OF OUABAIN AND METABOLIC INHIBITING FACTORS ON Ca DISTRIBUTION DURING K-INDUCED CONTRACTURE IN GUINEA PIG TAENIA COLI

It was reported that an application of high-K (isotonic, 40 mM) caused an increase in oxygen consumption accompanied by an increase in tension change in guinea pig taenia coli (1) and that these changes were reversibly abolished by a depletion of Ca from the high-K medium and Sr could replace Ca only for oxygen consumption (2). These results suggest that Ca entering smooth muscle treated with high-K probably increases oxygen consumption, independent of an increase in muscle tension. In a previous paper (3), effects of factors inhibiting tension development on oxygen consumption of the muscle in high-K medium were reported. Glucose removal from the high-K medium abolished the already developed tension corresponded with a decrease in the elevated oxygen consumption rate. Addition of DNP (1×10-4M) to high-K medium further increased or maintained the increased oxygen consumption although it abolished the already developed tension, and the change in oxygen consumption was not modified by Ca removal. On the other hand ouabain (2.5×10-6 M) maintained the increased oxygen consumption in high-K medium, but almost abolished the developed tension. The increase in oxygen consumption was dependent on external calcium. In the present paper, effects of factors inhibiting high-K induced tension and/or oxygen consumption on Ca distribution in taenia coli are studied and this throws some light on a participation of Ca distribution in changes in tension and oxygen consumption of the muscle in high-K medium. A study on ouabain and DNP with high-K on Ca exchange in taenia coli has been reported by Pfaffman and Holland (4), which is incompatible with the present data.

Role of protein synthesis in the inhibitory action of adrenal steroid hormones on amino acid transport by muscle.

Adrenal steroid hormones added in vitro inhibit the accumulation of amino acids by isolated rat diaphragm, but inhibition appears only some hours after the presumed interaction of the hormone with the tissue. Since it is known that the administration of corticosteroids to rats impairs protein synthesis in muscle, experiments were performed to explore the possibility that the delay in their effect on amino acid uptake might be due to the inhibition of protein synthesis. Preincubation of intact diaphragms of adrenalectomized rats with corticosterone (10-5M) for 2-3 hr did suppress the subsequent incorporation of L-methionine-14C into total diaphragm protein, although preincubation for 1 hr had no effect. This time course of inhibition was similar to that observed when AIB accumulation was measured. Preincubation of diaphragms with puromycin (0.37 HIM) or actinomycin D (5 μg/ml) inhibited the subsequent accumulation of AIB by the cells, and the time course of inhibition was comparable to that produced by corticosterone. When diaphragms were preincubated for 3 hr in medium containing both puromycin and corticosterone, AIB accumulation was inhibited, but to an extent equal to that produced by either substance alone. However, accumulation of AIB could be inhibited further by the addition of DNP (10-4M) to the medium. AIB accumulation was also suppressed in diaphragms isolated from rats that had received injections of the amino acid analogue, DLethionine. The agreement of these findings with the idea that corticosteroids inhibit muscle amino acid transport through their inhibitory action on protein synthesis is discussed. (Endocrinology79: 531, 1966)

OXIDATIVE PHOSPHORYLATION BY PEA SEEDLING MITOCHONDRIA

Using P32in combination with a procedure for quantitative separation of inorganic phosphorus a sensitive method for measuring phosphorylation was devised. It was employed to determine the effects of presoaking seed treatments, and of two inhibitors, 2,4-dinitrophenol (DNP) and malonate, on oxidative phosphorylation by pea seedling mitochondria. The addition of DNP to the reaction mixture resulted in typical uncoupling, but there was an unexpected increase in phosphorylation due to malonate. The length and weight of seedlings presoaked in aerated running water were higher than for those presoaked in standing water; however, mitochondrial preparations from seedlings presoaked in standing water gave higher phosphorylative and oxidative activities. These differences in results were regarded as evidence of 'physiological predetermination'.

Stimulation and inhibition of mitochondrial adenosine triphosphatase activity by oleate

1. 1. The release of the latent ATPase activity of fresh rat-liver mitochondria by sodium oleate was largely dependent on added Mg 2+ ions. The residual activity obtained in the absence of Mg 2+ ions was attributed to the presence of Mg 2+ ions in the mitochondria. 2. 2. Oleate inhibited the DNP-activated ATPase activity. (a) In the absence of Mg 2+ ions inhibition increased over the whole range of oleate concentrations tested (5–50 μg per 1.8 ml medium), 80–90% inhibition being obtained with 50 μg oleate. (b) In the presence of Mg 2+ ions a maximum inhibition of approx. 50% was obtained with about 25 μg oleate. At higher concentrations of oleate, however, the splitting of ATP was not inhibited or was even greater than that noted in the presence of Mg 2+ ions and DNP alone. This biphasic response is attributed to the release of the Mg 2+-dependent ATPase activity and the inhibition of the DNP-dependent ATPase activity, both effects progressing with increasing concentrations of oleate. 3. 3. Similar observations were made with mildly-aged liver mitochondria. The Mg 2+-dependent ATPase activity of liver mitochondria disrupted by sonic oscillation for 20 min was not influenced either by oleate or DNP, alone or in combination. 4. 4. When the medium was supplemented with the ADP-trapping system, phosphoenolpyruvate and pyruvate kinase, the Mg 2+-dependent ATPase activity of fresh liver mitochondria reacted towards increasing amounts of oleate in a manner similar or related to that shown by the DNP-dependent ATPase obtained in the absence of phosphoenolpyruvate and pyruvate kinase but with Mg 2+ ions present. After mild ageing of the particles and/or addition of DNP, and incubation in the presence of Mg 2+ ions, phosphoenolpyruvate and pyruvate kinase, the Mg 2+-dependent and/or DNP-dependent ATPases were first inhibited and then activated by increasing amounts of oleate.

Biochemical properties of skeletal muscle mitochondria: II. The ATPase activity and the albumin effect

The ATPase activity of freshly prepared pigeon muscle mitochondria is strongly stimulated by addition of Mg 2+ ions. This property is not peculiar to pigeon breast mitochondria, since muscle mitochondria prepared from hearl and skeletal muscles, whether from rat or pigeon, show a similar behavior. The mitochondrial ATPase of rat or pigeon liver mitochondria is not stimulated by Mg 2+. The pH activity curves for ATP hydrolysis indicate that the mitochondria prepared from liver, heart, and skeletal muscle of rat are strongly stimulated by addition of DNP both in presence and in absence of Mg 2+. In contrast the stimulating effect of DNP on mitochondria prepared from liver, heart, and breast of pigeons, is very small and not evident on the acid region of the curve. Bovine plasma albumin added to the medium does not change the oxidation rate of pigeon breast mitochondria, while it strongly increases the phosphorylation capacity and therefore the P O ratio. This effect is associated with an activation of the DNP-stimulated ATPase, particularly between pH 7.5 and 8.5. The effect of albumin on the DNP-stimulated ATPase is shown by all the muscle mitochondria examined and also by those of pigeon liver. Differently from DNP, addition of arsenate to pigeon breast mitochondria strongly stimulates the ATPase activity, whereas the P O ratio is only slightly affected.

Investigations of Ion Uptake by Tissue Slices II. The effect of 2,4‐diuitrophenol on the uptake of calcium hy potato slices

DNP (2,4‐dinitrophenol) has been found by a number of investigators to inhibit both anion and cation uptake in a variety of plant tissues (Arisz 1953, Hopkins 1956, Middleton 1955, Ordin and Jacobson 1955, Robertson et al. 1951, Russell and Ayland 1955). Following some preliminary investigations, Chasson and Levitt (1956) reported in a brief note that calcium uptake by potato slices was stimulated by the addition of DNP. Presented below are the results of a more thorough study of these initial observations.