scholarly journals The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1980 ◽  
Vol 192 (3) ◽  
pp. 821-828 ◽  
Author(s):  
David D. Tyler
Keyword(s):  
Atpase Activity ◽  
Large Amplitude ◽  
Atp Hydrolysis ◽  
Adenine Nucleotide ◽  
Sucrose Solution ◽  
Adenine Nucleotides ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Nucleotide Exchange ◽  
Major Activity

1. The distribution of Pi between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J.111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of Pi transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total Pi liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the Pi liberated was retained in the mitochondria. 2. Larger amounts of Pi appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this Pi were identified: (a) a slow efflux of Pi from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg2+, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and Pi and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and Pi, is unaffected by uncoupling agents and is activated by endogenous or added Mg2+. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the Pi liberated was recovered in the suspending medium, unless inhibitors of Pi-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg2+ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of Pi retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the Pi transporter provides the sole pathway for Pi efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg2+ on mersalyl inhibition of the Pi transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in Pi efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A.75, 4788–4792] are erroneous and should be discarded.

scholarly journals Deterioration of rat liver mitochondria under conditions of metabolite deprivation

1980 ◽  
Vol 188 (3) ◽  
pp. 817-822 ◽  
Author(s):  
J W Parce ◽  
P I Spach ◽  
C C Cunningham
Keyword(s):  
Atpase Activity ◽  
Sucrose Solution ◽  
Adenine Nucleotides ◽  
Respiratory Control ◽  
Liver Mitochondria ◽  
Phospholipid Metabolism ◽  
Rat Liver Mitochondria ◽  
Second Phase ◽  
Mitochondrial Atpase ◽  
Sequence Of Events

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.

scholarly journals The effect of butacaine on adenine nucleotide binding and translocation in rat liver mitochondria

1975 ◽  
Vol 148 (3) ◽  
pp. 527-531 ◽  
Author(s):  
D R Fayle ◽  
G J Barritt ◽  
F L Bygrave
Keyword(s):  
Rat Liver ◽  
Local Anaesthetic ◽  
Binding Sites ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Nucleotide Binding ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Inner Membrane

The effect of the local anaesthetic, butacaine, on adenine nucleotide binding and translocation in rat liver mitochondria partially depleted of their adenine nucleotide content was investigated. The range of butacaine concentrations that inhibit adenine nucleotide translocation and the extent of the inhibition are similar to the values obtained for native mitochondria. Butacaine does not alter either the total number of atractyloside-sensitive binding sites of depleted mitochondria, or the affinity of these sites for ADP or ATP under conditions where a partial inhibition of the rate of adenine nucleotide translocation is observed. The data are consistent with an effect of butacaine on the process by which adenine nucleotides are transported across the mitochondrial inner membrane rather than on the binding of adenine nucleotides to sites on the adenine nucleotide carrier. The results are briefly discussed in relation to the use of local anaesthetics in investigations of the mechanism of adenine nucleotide translocation.

scholarly journals Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

1978 ◽  
Vol 176 (3) ◽  
pp. 705-714 ◽  
Author(s):  
Veronica Prpić ◽  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Molecular Mechanisms ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Mitochondrial Protein ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
The Matrix

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca2+ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-Pi. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca2+ is retained for 6–8min. The ability of glucagon to enhance Ca2+ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca2+ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca2+ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca2+ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca2+ transport revealed a significantly higher concentration of adenine nucleotides but not of Pi in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by Pi treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca2+. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca2+-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

scholarly journals Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria

1974 ◽  
Vol 140 (3) ◽  
pp. 413-422 ◽  
Author(s):  
Terry L. Spencer ◽  
Fyfe L. Bygrave
Keyword(s):  
Rat Liver ◽  
Protein Interactions ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Local Anaesthetics ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Low Concentrations ◽  
High Concentrations ◽  
Lipid Protein Interactions

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid–protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30μm for 200μm-ATP and at 10μm for 200μm-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50μm) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250μm) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca2+ was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca2+. 4. The data provide further support for our hypothesis that lipid–protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.

scholarly journals The transport and accumulation of adenine nucleotides during mitochondrial biogenesis

1980 ◽  
Vol 192 (1) ◽  
pp. 75-83 ◽  
Author(s):  
J K Pollak ◽  
R Sutton
Keyword(s):  
Rat Liver ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Liver Mitochondria ◽  
Adenine Nucleotide Translocase ◽  
Luciferase Assay ◽  
Rat Liver Mitochondria ◽  
Matrix Space ◽  
Foetal Rat

The atractyloside-insensitive accumulation of adenine nucleotides by rat liver mitochondria (as opposed to the exchange-diffusion catalysed by the adenine nucleotide translocase) has been measured by using the luciferin/luciferase assay as well as by measuring [14C]ATP uptake. In foetal rat liver mitochondria ATP is accumulated more rapidly than ADP, whereas AMP is not taken up. The uptake of ATP occurs against a concentration gradient, and the rate of ATP uptake is greater in foetal than in adult rat liver mitochondria. The accumulated [14C]ATP is shown to be present within the mitochondrial matrix space and is freely available to the adenine nucleotide translocase for exchange with ATP present in the external medium. The uptake is specific for ATP and ADP and is not inhibited by adenosine 5′-[beta gamma-imido] triphosphate, GTP, CTP, cyclic AMP or Pi, whereas dATP and AMP do inhibit ATP accumulation. The ATP accumulation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, KCN and mersalyl but is insensitive to atractyloside. The ATP uptake is concentration-dependent and exhibits Michaelis-Menten kinetics. The divalent cations Mg2+ and Ca2+ greatly enhance ATP accumulation, and the presence of hexokinase inhibits the uptake of ATP by foetal rat liver mitochondria. These latter effects provide an explanation for the low adenine nucleotide content of foetal rat liver mitochondria and the rapid increase that occurs in the mitochondrial adenine nucleotide concentration in vivo immediately after birth.

Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Pi carrier

2003 ◽  
Vol 285 (2) ◽  
pp. G274-G281 ◽  
Author(s):  
Thilo Hagen ◽  
Christopher J. Lagace ◽  
Josephine S. Modica-Napolitano ◽  
June R. Aprille
Keyword(s):  
Rat Liver ◽  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Permeability ◽  
Nucleotide Content ◽  
Adenine Nucleotide Pool

Mitochondrial permeability transition, due to opening of the permeability transition pore (PTP), is triggered by Ca2+ in conjunction with an inducing agent such as phosphate. However, incubation of rat liver mitochondria in the presence of low micromolar concentrations of Ca2+ and millimolar concentrations of phosphate is known to also cause net efflux of matrix adenine nucleotides via the ATP-Mg/Pi carrier. This raises the possibility that adenine nucleotide depletion through this mechanism contributes to mitochondrial permeability transition. Results of this study show that phosphate-induced opening of the mitochondrial PTP is, at least in part, secondary to depletion of the intramitochondrial adenine nucleotide content via the ATP-Mg/Pi carrier. Delaying net adenine nucleotide efflux from mitochondria also delays the onset of phosphate-induced PTP opening. Moreover, mitochondria that are depleted of matrix adenine nucleotides via the ATP-Mg/Pi carrier show highly increased susceptibility to swelling induced by high Ca2+ concentration, atractyloside, and the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Pi carrier, by regulating the matrix adenine nucleotide content, can modulate the sensitivity of rat liver mitochondria to undergo permeability transition. This has important implications for hepatocytes under cellular conditions in which the intramitochondrial adenine nucleotide pool size is depleted, such as in hypoxia or ischemia, or during reperfusion when the mitochondria are exposed to increased oxidative stress.

scholarly journals Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1974 ◽  
Vol 142 (2) ◽  
pp. 353-358 ◽  
Author(s):  
Susan M. Danks ◽  
J. B. Chappell
Keyword(s):  
Citrate Synthase ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Glycerol Phosphate ◽  
Matrix Space ◽  
Carbonyl Cyanide ◽  
The Matrix ◽  
Metabolic Conditions

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD+-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.

P1, P5-Bis-(5′-adenosyl)pentaphosphate: Is this Adenylate Kinase Inhibitor Substrate for Mitochondrial Processes?

1977 ◽  
Vol 32 (9-10) ◽  
pp. 786-791 ◽  
Author(s):  
Josef Köhrle ◽  
Joachim Lüstorff ◽  
Eckhard Schlimme
Keyword(s):  
Adenylate Kinase ◽  
Kinase Inhibitor ◽  
Adenine Nucleotide ◽  
Nucleoside Diphosphate Kinase ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Rabbit Muscle ◽  
Inner Mitochondrial Membrane

Abstract 1. P1, P5-Bis-(5′-adenosyl)pentaphosphate (Ap5A) inhibits “soluble” adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10-5м , i. e. about two orders of magnitude higher than the inhibitor constants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme.2. As to be expected Ap5A which is of the “multisubstrate analogue”-type does not affect mito­ chondrial nucleoside diphosphate kinase.3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor specifically bound.4. Ap5A is not metabolized by rat liver mitochondria.

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