ATP Resynthesis Research Articles

Prolonged depletion of ATP and of the adenine nucleotide pool due to delayed resynthesis of adenine nucleotides following reversible myocardial ischemic injury in dogs

After 15 min of severe ischemia induced by circumflex artery occlusion in open-chest dogs, 65% of the ATP and 50% of the total adenine nucleotide (ΣAd) pool is lost from the subendocardial myocardium [ 12]. Nevertheless, this injury is reversible if the affected tissue is reperfused with coronary arterial blood. In the present experiment, we assessed the effects of various periods of arterial reflow following 15 min of ischemic injury, on resynthesis of ATP and ΣAd. The circumflex artery was occluded for 15 min and reperfused for 20 or 60 min, or 24 or 96 h. Ten seconds prior to excision of the heart, the circumflex artery was reoccluded and the fluorescent dye thioflavine S was injected intravenously in order to identify the ischemic or the reperfused tissue which had been ischemic. The mean ATP after 15 min of ischemia was reduced 62% from 5.42 ± 0.33 to 2.08 ± 0.21 μmol/g; and the total nucleotide content was reduced by 50%. ATP content recovered slightly during the first 20 min of reperfusion but remained markedly depressed for at least 24 h because of the initial depletion of adenine nucleotides and because minimal salvage or de novo synthesis occurred in the injured muscle during this time period. By 4 days, ATP and total adenine nucleotides were still slightly depressed but had recovered to 88% and 91% of control. Electrolyte changes and an increased inulin diffusible space, which are characteristic of irreversibly injured myocardium reperfused for 20 or 60 min, were not observed. Also tissue necrosis was absent in the hearts reperfused for 24 or 96 h. These observations indicate that the marked depression of ATP and adenine nucleotides and the slow recovery of these metabolites occurred in myocardium which nevertheless was reversibly injured in terms of cellular viability.

A protective effect of coenzyme Q 10 on ischemia and reperfusion of the isolated perfused rat heart

Experiments were undertaken to determine if pretreating the animal with coenzyme Q 10 (CoQ) protected the cardiac muscle of the isolated perfused heart which was exposed to global ischemia for 30 min and then reperfusion. Two hours after the intramuscular (20 mg/kg) and intraperitoneal (10 mg/kg) injections of CoQ or vehicle alone the hearts were excised and perfused by Langendorff's technique. In the vehicle-pretreated group, ischemia caused a precipitous decline of the contractile tension development, an elevation of the resting tension and a severe contracture. During the substrate-free reperfusion for 30 min, there was a small decrease in the magnitude (25%) of the contracture, and, yet, no tension development. In the additional reperfusion with glucose as the substrate for 30 min, the tension development was slightly (11%) recovered. Tissue ATP and total adenine nucleotide contents at the end of the glucosereperfusion markedly decreased. Although pretreatment with CoQ had no effect on the onset and progression of ischemic contracture, it facilitated the recovery of mechanical performance. Cardiac stores of both ATP and total adenine nucleotide in CoQ-pretreated reperfused hearts were significantly ( P < 0.01) higher than those in vehicle-pretreated group. There was a direct relation between the extent of the recovery of mechanical performance and tissue ATP content. It was suggested that exogenous CoQ protected the cardiac muscle from the deterioration of mechanical function of ischemia and recovery. Better mechanical function of CoQ-pretreated heart was attributable to better resynthesis of ATP presumably due to lesser loss of adenine nucleotide pool from the cardiac cells.

Adenine nucleotide metabolism during hepatic ischemia and subsequent blood reflow periods and its relation to organ viability.

In an experimental model system for liver transplantation, the ability of the rat liver to synthesize ATP and to maintain adequate energy charge and total adenine nucleotides during restoration of hepatic blood flow following warm ischemia was found to determine tissue viability and survival of the animal. A portafemoral shunt prepared to relieve portal congestion enhanced the rate and extent of ATP resynthesis by the reflow following hepatic ischemia and this was accompanied by an increase in the survival rate of the rat.

Oxidative phosphorylation in rat skin during preservation

The addition of sodium dihydrogen orthophosphate to storage buffers results in markedly higher intracellular ATP concentrations in rat skin stored at −196 and −3 °C as compared to storage in phosphate-free medium. The inorganic and total phosphorus depletion occurring in phosphatefree buffers can be compensated for by the addition of orthophosphates to the storage medium. The stimulatory effect of orthophosphate on the metabolic activity of stored tissue is attributed to an effective protection of oxidative phosphorylation. This is achieved by providing for the essential phosphate compounds necessary for constant resynthesis of ATP.

The role of coenzyme Q 10 for preservation of the rat kidney: A model experiment for kidney transplantation

In an experimental model system for kidney transplantation, the ability of the rat kidney to resynthesize ATP after warm ischemic time and subsequent restoration of renal blood flow was found to determine tissue viability and survival of the animal. The administration of CoQ 10 prior to warm ischemia enhanced the rate and extent of ATP resynthesis by reflow following two-hour warm ischemia and this was accompanied by an increase in survival rate of the rats.

Chemical change and energy production during contraction of frog muscle: how are their time courses related?

1. The heat+work (h+w) and the changes in the levels of creatine, phosphocreatine and ATP were determined for 1, 2, 5, 10 and 15 sec isometric tetani at 0 degrees C under aerobic conditions. The change in the sum of inorganic phosphate and glucose-1-phosphate and glucose-6-phosphate was measured also. The changes in the levels of all these chemicals and of lactate were measured in muscles stimulated for 15 sec under anaerobic conditions. 2. The lactate measurements and a comparison of the results for aerobic and anaerobic conditions showed that during a 15 sec tetanus there was a negligible amount of resynthesis of ATP from reactions other than the creatine kinase reaction. 3. For all durations of stimulation, except 1 sec, a significant part of the h+w could not be explained by the energy from ATP splitting and the creatine kinase reaction. The existence of an unexplained part of the h+w confirms earlier findings. 4. On the basis of its time course, the h+w was divided into the stable part and the labile part. The energy from the observed chemical reactions was always sufficient to account for the stable part of the h+w. 5. Early in the tetanus the unexplained energy is less than the labile part of the h+w. At the end of a 15 sec tetanus the total amounts of unexplained energy and labile h+w are equal. For this reason and others which are discussed it is probable that there is a close relationship between them.

The effects of adenosine triphosphate and inorganic phosphates on the viability of stored rat skin

Additions of ATP and inorganic phosphates to storage buffers, increase the viability of rat skin when stored at −196 °C and at −3 °C. The amino acid incorporation into the skin proteins, the α-[1- 14C]-aminoisobutyric acid uptake by the skin and to a lesser extend the [6- 3H]thymidine incorporation into DNA are protected by the phosphate compounds added to the storage medium. This stimulatory effect on the metabolic activity appears connected with the preservation and the protection of the oxidative phosphorylation, probably by providing the necessary phosphate radicals for the resynthesis of ATP. By simultaneously preventing potassium depletion during cooling and storage, the potassium phosphate compounds seem particularly suited to contribute to the preservation of the viability.

Chemical energetics of single isometric tetani in mammalian smooth muscle.

A rabbit taenia coli preparation has been used to study the chemical energetics of smooth muscle contraction. Under the experimental conditions, the muscle had no spontaneous mechanical activity, but could be fully activated with the use of electrical field stimulation. ATP resynthesis from glycolysis and respiration was stopped with a procedure that involved treatment with metabolic inhibitors at 5 degrees C followed by rewarming to 18 degrees C. This procedure did not alter the high-energy phosphate contents of mechanical responses of the muscle. Use of this preparation to determine ATP and phosphorylcreatine changes during isometric tetani at 18 degrees C and a length of 86% LO showed that there was net ATP breakdown initially with no significant phosphorylcreatine splitting. This was followed by an increase in the rate of phosphorylcreatine splitting with net ATP synthesis. The average rate of total high-energy phosphate utilization was about 0.01 mumol/g.s for up to 60 s or about 150 times less than that of frog sartorius at the same temperature.

The role of glucose and glycogen as fuels for muscle.

During muscular work, chemical energy is converted into mechanical energy. The site of this energy exchange, which involves the biological energy currency ATP, is the cross-bridges between the contractile proteins actin and myosin, and the energy is used during the breaking and reattachment cycles of the cross-bridges. The terminal phosphate group and ADP moiety of ATP are transferred to the participating systems in the contractile system, and eventually Pi and ADP are released so that, when the net reaction is completed, ATP has been hydrolysed. The resynthesis of ATP from ADP and Pi occurs from some of the energy made available in the oxidative reactions of the cell. Consequently, the energy conserved during oxidation is transferred to the processes that utilize energy (e.g. muscular contraction) via the ATP/ADP-coupled system in Scheme 1. It cannot be emphasized too strongly that ATP is not stored in the cell; its concentration in muscle is only 5-7m~ (see Beis & Newsholme, 1975), which would be depleted in less than a second during contraction unless it is resynthesized. Furthermore, to maintain the steady-state concentration of ATP, the increase in the rate of resynthesis must equal precisely that of utilization (see Newsholme & Start, 1973). That this occurs is demonstrated by the many observations that the ATP concentration remains remarkably constant despite very large variations in the power output of a given muscle. These observations strongly suggest the existence of specific mechanisms that regulate the rate of ATP formation to that of its utilization. These mechanisms must be sensitive, precise and permit the rapid transfer of information between the utilizing and

Splanchnic organ adenine nucleotides and their netabolites in haemorrhagic shock.

The concentrations of ATP, ADP and AMP in splanchnic organs, and the plasma concentrations of the end products of adenine nucleotide breakdown, were determined in experimental haemorrhagic shock. Haemorrhagic hypotension resulted in marked reduction in the ATP and the total adenine nuclectide contents of pancreas, liver and duodenum even two hours following reinfusion. Plasma hypoxanthine and uric acid increased more than ten fold during the hypotensive period, and later decreased gradually. The plasma concentration of allantoin increased dramatically during haemorrhagic hypotension and did not decrease following reinfusion. It is proposed that when breakdown of adenine nucleotides in haemorrhagic hypotension proceeds as far as uric acid and allantoin, the resynthesis of ATP following reinfusion is severely limited. The possibility that this disturbance in energy metabolism may influence the irreversibility of haemorrhagic shock is discussed. with particular reference to the pancreas.

The physical state of water and ions in living cells and a new theory of the energization of biological work performance by ATP.

In this article, the key concepts of the association-induction hypothesis and their experimental verifications were reviewed: According to this hypothesis, the bulk of cell water exists in the state of multilayers polarized and oriented by the backbones of certain proteins existing in an extended state. The major intracellular cation, K+, is also adsorbed but singly and on different protein sites (i.e.,β &γ-carboxyl groups). The living protoplasm of protein, ion and water represents a three-dimensional cooperative assembly under the control of certain cardinal adsorbents, of which ATP is a prime example. Association of ATP with the cardinal site of a key protein may produce a cooperative change in the electron distribution of the protein with consequent change of the state of ion and water adsorption. Such an alteration in the physical state of water leads to a change in the solubility of various solutes in this water as well as permeability of various solutes (e.g., Na) through this water. In contrast to the conventional concept, the role of ATP is not to provide a package of energy held in a special chemical bond. Rather, as a cardinal adsorbent, ATP acts by means of its specific electronic interaction with the protein molecules, maintaining the protein—ion—water system at a higher energy state. Dephosphorylation of ATP leads to a cooperative shift of the electron state of the whole assembly to a lower energy state. Restoration to the original resting state follows the resynthesis of ATP (where energy is injected into the system) and its readsorption on the cardinal site.

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.

Inhibitory effect of D-glucosamine on glycolysis in bovine retina.

1. The effects of glucosamine concentration on the size of the lactate pool, on the levels of ATP, ADP, AMP and on the radioactivity incorporation from [1-14-C] glucosamine into lactate, N-acetylglucosamine and glucosamine-6-P were studied using whole bovine retinas. 2. The radioactive lactate, evaluated in relation to glucosamine molarity, after a modest initial increase, diminishes significantly. On the contrary the N-acetyl [1-14-C] glucosamine, the [1-14-C] glucosamine-6-P and, consequently, also the [1-14-C] glucosamine-6-P/[-14-C] lactate ratio increase with glucosamine molarity. 3. The retinal content of ATP shows a modest increment after incubation with low concentrations of D-glucosamine (0.5--2.0 mM) and a remarkable fall at higher concentrations. 4. Using retinal homogenates D-glucosamine clearly lowers the lactate production from glucose, glucose-6-P and fructose-1, 6-P2. 5. D-Glucosamine acts as an inhibitor of retinal glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase by decreasing the initial velocity of these reactions. 6. It is concluded that D-glucosamine causes a reduction in the lactate production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase. The fall in the adenine nucleotides content is a consequence of a dephosphorylation of ATP for the phosphorylation of glucosamine without concomitant resynthesis of ATP "via glycolysis".

Effect of uracil analogues on the oxidative and phosphorylating function of albino rat liver mitochondria

After intraperitoneal injection of 4-methyluracil (4MU) and 4-methyl-5-hydroxy-uracil (4M5HU) for four days in doses of 100 mg/kg daily partial inhibition of malate- and succinate-dehydrogenase activity was found in the liver mitochondria. The utilization of inorganic phosphate for ATP resynthesis was inhibited, and this was evidently one cause of the decrease in the ATP content in the liver. Administration of 4M5HU in a dose of 30 mg/kg was accompanied by similar changes but they were less marked. Under the influence of 4 MU in a dose of 30 mg/kg only the rate of phosphorylation in the mitochondria and the ATP content in the liver were significantly changed (increased).

Apparent [formula omitted] ratio and chemical energy balance in frog sartorius muscle in vitro

We tested the hypothesis that there is a constant relation between the amount of chemical energy used during a single tetanus and the extent of subsequent aerobic recovery metabolism. Breakdown of high energy compounds (Δ ∼ P) during contraction was measured by rapid freezing techniques using isometric contractions of unpoisoned frog sartorius muscles at 0 °C. A stable and sensitive method was designed to study recovery O 2 consumption of similar contractions. In some cases, initial chemical changes and recovery O 2 consumption were measured in the same muscle. The ratio of these chemical changes yields a value for the P/O ratio of whole muscle. This ratio was found to be 2 and was constant for the range of contraction durations studied (5–20 s). Splitting of phosphorylcreatine or ATP after mechanical relaxation and glycolytic resynthesis of ATP could not be detected. Thus, the tested hypothesis is valid; but there is no ready explanation why this estimate of the P/O ratio of whole muscle differs from the ratio measured in isolated mitochondria. Because of this constancy and the observed stoichiometry unknown energy yielding reactions, postulated to occur either early or late in a maintained tetanus, are excluded by these experiments.

Actin as an energy transducer

When active muscle is stretched, the energy-yielding process for contraction is reversed. In this “reversed contraction”, work is expanded to resynthesize ATP and thus in this process muscle may be classed with mitochondria and chloroplasts. It is argued that actin, the main constituent of the thin filaments, is analogous to the coupling factors of mitochondria and chloroplasts where ATP resynthesis takes place.

Regulation of Glyceraldehyde 3-Phosphate Dehydrogenase by Phosphocreatine and Adenosine Triphosphate: IV. FACTORS AFFECTING IN VIVO CONTROL OF ENZYMATIC ACTIVITY

Abstract Phosphocreatine inhibited glyceraldehyde 3-phosphate dehydrogenase crystallized from rabbit muscle or yeast. Creatine at the same concentration showed no inhibition. The inhibition was competitive with respect to glyceraldehyde 3-phosphate. At the approximate physiological concentrations of substrate (0.01 mm) and phosphocreatine (20 mm), the enzyme was 65 % inhibited. Phosphocreatine is not competitive with respect to NAD or orthophosphate. The inhibition by phosphocreatine was maximal at pH 7.4 and decreased about 17 % with a pH fall to 6.8 or a rise to 8.5. Incubation of the muscle enzyme with phosphocreatine produced a time-dependent inactivation which was prevented by the addition of glyceraldehyde 3-phosphate. This time-dependent inactivation and the instantaneous inhibition were additive. Magnesium partially relieved the inhibitory effects of phosphocreatine. The properties of the inhibitory effects of phosphocreatine and ATP are compared. Since the phosphocreatine level in muscle may decrease very rapidly during muscle contraction, the inhibition on triose phosphate dehydrogenase may be lessened substantially, and ATP resynthesis will be promoted. Further physiological consequences of phosphocreatine and ATP inhibition in the resting and contracting muscle are considered.

Selective in vitro cytotoxicity of a glycolytic enzyme inhibitor for immature lymphoid cells.

Summary. A specific lactate dehydrogenase inhibitor, sodium oxamate, which prevents glycolytic resynthesis of ATP, has been shown to possess a significant, in vitro cytotoxic effect for EB2 cells and for the acute leukaemia lymphoblast. This result was obtained at an oxamate concentration of 0.08 M in TC 199 medium adjusted to physiological osmolality, in comparison with control cultures containing an equimolar concentration of sodium chloride. No significant oxamate cytotoxicity was found for normal marrow myeloid precursors or for blood lymphocytes and neutrophils, although a significant reduction in glycolysis‐dependent neutrophil phagocytosis was obtained.

Studies on the mechanism of the inhibition of protein synthesis induced by intracellular ATP depletion

The deleterious effect of intracellular ATP depletion on the protein-synthesizing system was studied in rabbit reticulocytes and Landschütz ascites tumor cells. The progressive decline of the ATP content of the cells induced by anaerobic incubation in glucose-free Krebs-Ringer salt solution was attended by a prompt inhibition of protein synthesis. The onset of the arrest of protein synthesis in the course of incubation under nitrogen, coincident with a 20–30 % decrease of the ATP level, entailed no impairment of the cell-free amino acid-incorporating activity and was readily reversible by reaeration of the cells. This early inhibition showed no correlation with the degree of ATP deprivation but appeared to be determined by the inhibitory effect of the accumulating ADP and AMP on the process of peptide chain elongation. Extensive ATP depletion following prolonged anaerobic incubation gave rise to a lesion characterized by dissociation of the polysomes and a concomitant decline of the cell-free amino acid-incorporating activity. However, the expression of this lesion was masked by factors interfering with the peptide chain elongation. Therefore, drastic dissociation of the polysomes within the cells occurred only during the brief anaerobic-aerobic transition period following readmission of air when the block of the chain elongation by the products of ATP splitting had been relieved by partial resynthesis of ATP. On prolonging the aerobic reincubation of the cells, progressive reversal of the derangement of protein synthesis took place. It was concluded that the lesion concerned was localized at the stage of peptide initiation. The initiation defect caused by severe ATP deficiency was shown to reside exclusively in the cell-sap fraction and to differ essentially from the ribosomal lesion induced by NaF. In addition, the detrimental effect of NaF on protein synthesis proved to be entirely unrelated to the inhibitory action of this poison on the energy metabolism. An inhibitory pattern essentially similar to the one above was obtained when ATP depletion was brought about by incubating the cells in the presence of 2,4-dinitrophenol. Furthermore, the alterations induced by the uncoupling action of dinitrophenol on the oxidative phosphorylation were completely reversible by washing away the uncoupling agent. The mechanism underlying the above alterations of the protein-synthesizing system are discussed in light of the pertinent data, and the possible regulatory implication of the inhibitory influence of the products of ATP breakdown on peptide chain elongation is considered.

ATP requirement for methanogenesis in cell extracts of methanobacterium strain M.o.H.

Abstract 1. 1. ATP stimulation of methane formation in cell extracts of hydrogen-grown Methanobacterium strain M.o.H. has been studied. 2. 2. Less than one ATP molecule is broken down per molecule of methane produced. 3. 3. Resynthesis of ATP by the cell-free extract is to show to account for this lack of stoichiometry. 4. 4.Wnen cell-free extracts are producing methane at a maximal rate after ATP addition, the depletion of ATP by a hexokinase trap does not stop methane synthesis.