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1.
The dependence of both respiration and total activity of ATP-consuming reactions on the cellular adenine nucleotide pattern was investigated in intact bovine spermatozoa. ATP consumption was manipulated by inhibition with vanadate and activation with caffeine, leading to a decrease or increase in the rate of respiration up to 70% or 20%, respectively. Oligomycin blocked the respiration to the same extent as did vanadate, suggesting that the total extramitochondrial ATP-consuming activity is vanadate-sensitive. The major part of ATP utilization must be linked to dynein ATPase, since inhibition of (Na +, K +) ATPase by ouabain showed only a small effect on respiration (−17%). Being a potent inhibitor of dynein ATPase, vanadate drastically reduced the amount of motile cells, whereas caffeine tended to increase the intensity of motion. The effects of vanadate or caffeine on respiration were paralleled by changes in cellular ATP, reflecting the response of mitochondrial respiration on the cellular ATP/ADP ratio. Respiration was found to depend on changes in the ATP/ADP ratio in the range from about 3 (+ caffeine) to 9 (+ vanadate). The range of response of ATP consumption to the ATP/ADP ratio was determined by varying the mitochondrial ATP production via the concentration of lactate which was used as substrate. The measured effects on both respiratory rate and ATP/ADP ratio suggested that ATP consumption was markedly dependent on ATP/ADP ratios below 5. It is concluded that lactate concentrations above 1 mM sufficiently supply bovine spermatozoa with substrate and the energy turnover is mainly limited by the activity of dynein ATPase rather than by the capacity of mitochondrial oxidative phosphorylation. 相似文献
2.
The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria. 相似文献
3.
Mitochondria from rabbit reticulocytes contain about 50% of the total reticulocyte hexokinases. The proportion of mitochondrial hexokinases may be changed under different metabolic conditions. Mitochondrial bound and soluble hexokinases exhibit different kinetic properties (KMATP and glucose-6-phosphate inhibition). The respiratory rate of isolated reticulocyte mitochondria in the presence of glucose depends on the glucose-6-phosphate concentration, as the ADP generation by the endogenous hexokinases is strongly inhibited by glucose-6-phosphate. In the experimental system all intermediary states of mitochondrial respiration can be adjusted between the state of maximal activity (state 3 or active state) and the controlled or resting state (state 4) by different glucose-6-phosphate levels. The stationary levels of the extramitochondrial adenine nucleotides in this experimental system have been measured. The rate of mitochondrial respiration and ATP formation depends on the extramitochondrial ATP/ADP ratio. At ratios of about 10 and lower the mitochondria are in their maximum phosphorylation state, at higher ratios the mitochondrial ATP formation is controlled by the extramitochondrial ATP/ADP ratio. It is postulated that the close intercounnection between the mitochondrial hexokinase and the mitochondrial ATP forming system in reticulocytes is of funcitonal significance for mitochondrial-cytosolic interactions in rabbit reticulocytes and probably in other types of cells with mitochondrial hexokinases, too. 相似文献
4.
Oligomycin is an inhibitor of the mitochondrial ATP synthase. In nitrogen-replete cells of the marine diatom Phaeodactylum tricornutum Bohlin, the rate of dark respiration was high and markedly inhibited (62%–74%) in the presence of oligomycin. In contrast, the rate of dark respiration in nitrogen-deprived cells was about half that in nitrogen-replete cells but was only slightly inhibited (16%–30%) by oligomycin. Consistent with these effects on rates of dark respiration, oligomycin decreased the ATP level and the ATP:ADP ratio by about 40% in nitrogen-replete cells incubated in darkness but had a negligible effect on the ATP level and ATP:ADP ratio in nitrogen-deprived cells. In sodium and nitrogen-deprived cells, the rate of dark respiration was greater than that in nitrogen-replete cells, but there was little effect of oligomycin on the rate of dark respiration. In light-limited cells, the rate of dark respiration was similar to that in nitrogen-deprived cells, but the inhibition (57%) in the presence of oligomycin was greater. These results suggest that most of the O 2 consumption by nitrogen-replete cells was linked to mitochondrial ATP synthesis and that the rate of mitochondrial ATP synthesis in nitrogen-deprived and sodium and nitrogen-deprived cells was low. The potential implications of these results for our understanding of maintenance respiration are discussed. 相似文献
5.
Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria. 相似文献
6.
The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
The efficiency of stimulation of mitochondrial respiration in permeabilized muscle cells by ADP produced at different intracellular sites, e.g. cytosolic or mitochondrial intermembrane space, was evaluated in wild-type and creatine kinase (CK)-deficient mice. To activate respiration by endogenous production of ADP in permeabilized cells, ATP was added either alone or together with creatine. In cardiac fibers, while ATP alone activated respiration to half of the maximal rate, creatine plus ATP increased the respiratory rate up to its maximum. To find out whether the stimulation by creatine is a consequence of extramitochondrial [ADP] increase, or whether it directly correlates with ADP generation by mitochondrial CK in the mitochondrial intermembrane space, an exogenous ADP-trap system was added to rephosphorylate all cytosolic ADP. Under these conditions, creatine plus ATP still increased the respiration rate by 2.5 times, compared with ATP alone, for the same extramitochondrial [ADP] of 14 microM. Moreover, this stimulatory effect of creatine, observed in wild-type cardiac fibers disappeared in mitochondrial CK deficient, but not in cytosolic CK-deficient muscle. It is concluded that respiration rates can be dissociated from cytosolic [ADP], and ADP generated by mitochondrial CK is an important regulator of oxidative phosphorylation. 相似文献
8.
1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio. 相似文献
9.
To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space. 相似文献
10.
Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP- P(NH) P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP- P(NH) P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria. 相似文献
11.
Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP(+)), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis. 相似文献
12.
The effect of TP (triosephosphates:glyceraldehyde-3 phosphate, GAP, +dihydroxyacetone phosphate, DHAP) on respiration, phosphorylation and matrix ATP/ADP ratios of isolated oat mesophyll mitochondria was investigated. With both malate and NADH, a 50% inhibition of state 3-phosphorylation was induced by about 15 to 20 millimolar GAP and 30 to 40 millimolar DHAP. However, the nature of the inhibition appeared to be different with the two respiratory substrates. In the presence of NADH, TP did not inhibit the rate of state 3 (addition of ADP) O 2 consumption. In fact, depending on concentration, TP gradually increased the rates measured without ADP towards those seen under state 3, acting as uncouplers. When malate was the substrate for respiration, state 3 rates were decreased. The effect was comparable to that of rotenone and could be abolished by the addition of NADH. These observations indicate a dual action of TP: inhibition of electron transport around site I and uncoupling. In any case, the intramitochondrial ATP/ADP ratio decreased upon addition of TP. The effective TP concentrations as well as the changes in mitochondrial ATP/ADP ratios were comparable to results on changes of compartmental pool sizes of adenylates and other metabolites during dark/light transition of oat mesophyll protoplasts (R. Hampp, M. Goller, H. Füllgraf, and I. Eberle 1985 Plant Cell Physiol 24: 99). The possible role of TP in the regulation of mitochondrial respiration in the light, as well as modes of interference, are discussed. 相似文献
13.
1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase. 相似文献
14.
Blowfly ( Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide- p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP ( Km = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent KI was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD +-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle. 相似文献
15.
Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production using antimycin A (AA) (complex III inhibition) combined with inhibition of ADP/ATP exchange by bongkrekic acid (BA) (adenine nucleotide translocator (ANT) inhibition) induced a sharp decrease in total cellular ATP in FL5.12 parental cells (to 35% of untreated controls after 24 h of incubation). Within 24 and 48 h, 38% and 75% of the cells had died, respectively. However, in stably transfected FL5.12 Bcl-2 subclones, no cell death occurred under these experimental conditions. Similar results were obtained with Jurkat and Bcl-2 overexpressing Jurkat cells. Total cellular ATP levels were equally affected in FL5.12 Bcl-2 overexpressing cells and FL5.12 parental cells. This indicates that Bcl-2 overexpressing cells are able to survive with very low cellular ATP content. Furthermore, Bcl-2 did not protect against cell death by restoring ATP levels. This suggests that, under these conditions, Bcl-2 acts by inhibiting the signalling cascade triggered by the inhibitors that would normally lead to apoptosis. 相似文献
16.
TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics. 相似文献
17.
The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios. 相似文献
18.
The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L). 相似文献
19.
The inhibitory effect of oligomycin was investigated in intact mitochondria through oxidative phosphorylation and uncoupler induced ATPase activity. Results show that oligomycin inhibition curves can be either sigmoidal or hyperbolic depending on experimental conditions and chiefly on the metabolic state of mitochondria with regard to the distribution of mitochondrial endogenous adenine-nucleotides. Active respiration and uncoupler-induced ATPse activity produce sigmoidal titration curves for a high initial ATP : ADP ratio and hyperbolic curves for a low ATP : ADP ratio. Time-dependent inhibitions are observed for the two reactions. The maximal inhibitory action for low concentrations of the inhibitor is delayed by the initial presence of ATP or the possibility of generating from inorganic phosphate before adding oligomycin. Results presented here show that the initial adenine-nucleotide distribution is important for oligomycin sensitivity of energy-linked reactions. Although a limited conformational change of the oligomycin-sensitivity to the inhibitor, it is more likely that a gross structural change of the inner membrane induced by adenine-nucleotides modifies membrane permeability to oligomycin. 相似文献
20.
Changes were measured in glycolytic and respiratory rates during the entire period of glycolysis and respiratory inhibition after addition of 0.08 or 0.15 mM glucose to Ehrlich ascites carcinoma cells in 54 mM phosphate buffer (pH 7.3) at 37°. Glycolytic products fully accounted for the glucose utilized. Theoretical rates of glycolytic ATP synthesis were calculated from the rates of accumulation of glycolytic products, and rates of oxidative phosphorylation were calculated from respiratory rates, assuming a P:O ratio of 3.0. The maximum in the glycolytic phosphorylation rate curve preceded the minimum in the respiratory phosphorylation rate curve. As a consequence, the total phosphorylation rate curve was biphasic, first rising above, then falling below, and finally returning to the initial, pre-glucose rate. The area under the early rise approximately equalled the area above the later dip and corresponded to between 1 and 2 μmoles of ATP/ml cells. The low rate of change in the ATP content of the cells indicated that most of the change in phosphorylation rate represented changes in both ATP synthesis and ATP utilization. It is hypothesized that ATP synthesized by glycolysis is more readily available to the ATP-utilizing systems. On addition of glucose, ATP is shifted from a respiratory to a glycolytic reservoir and a period of more rapid ATP utilization associated with a decrease in the level of endogenous substrates involved in the ATP-utilizing reactions ensues; after cessation of glycolysis, the process is reversed, and ATP utilization is slowed for a period while the endogenous substrates increase again. 相似文献
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