Adenine nucleotide transporters in organelles: novel genes and functions

In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.     

Analysis of nucleotides bound to the ATP synthetase from spinach chloroplasts isolated under different conditions

Summary The total amount of bound adenine nucleotides in the coupling factor isolated from spinach chloroplasts and its distribution on AMP, ADP and ATP was analyzed after various incubation conditions. During purification of the coupling factor, the distribution of AMP, ADP and ATP is not altered. The coupling factor from deenergized membranes contains approximately 1 ADP, less than 1 ATP, and small amounts of AMP. During phosphorylation the pattern is changed and ATP becomes the dominant species. When exogenous ADP is lacking, phosphate is readily incorporated into ATP. Inhibition of adenylate kinase by AP₅A does not change the distribution pattern of the adenine nucleotides. The distribution pattern shows no integer numbers for the different nucleotides, suggesting that the coupling factor is present in different states in a statistical distribution.Acknowledgment: We thank the Swiss National Foundation for Scientific Research (grant 3.582.79) for generous support.     

A comparison of the effects of the optical isomers of isoproterenol on energy metabolism in a mouse sarcoma

Disturbance to energy production in the S180 sarcoma (CB) by optical isomers of isoproterenol was assessed from altered adenine nucleotide levels at 1 h. The L-isomer almost halved the ATP level and lowered the energy charge significantly; the D-isomer was inactive. Dependence of tumor injury on cytochrome P-450 activity appears unlikely.     

Adenine nucleotide metabolism during anoxia and postanoxic recovery in insects

Severe hypoxia (anoxia), if maintained for more than a few minutes, causes irreversible damage in humans and other mammals. Why mammals are so vulnerable to anoxia is not fully understood. It is therefore of interest to study animals that are more tolerant of anoxia in order to identify physiological and metabolic properties that are correlated with a high tolerance of anoxia. Insects have high metabolic rates and their energy metabolism is dependent on aerobic ATP production. In insects, as in mammals, anoxia causes a rapid breakdown of physiological function, resulting in a state similar to rigor mortis. This is accompanied by a precipitous decrease in metabolic rate. In contrast to mammals, however, insects can survive anoxia for many hours and recover spontaneously and completely when air is again available. We have followed the metabolism of adenine nucleotides in locust tissues (mainly in the flight muscle) over 3 h of anoxia and during recovery from 1 h of anoxia. The content of ATP in the flight muscle dropped to 1% of normal during 2 h of anoxia. The main product was AMP which increased in content more than 20-fold. Some of the AMP was deaminated to IMP and this was further dephosphorylated to inosine. Altogether less than 30% of the total adenine nucleotides were degraded during 3 h of anoxia and this may contribute to the amazing ability of insects to recover from prolonged anoxia.     

Platelets, endothelium and blood vessel wall

Aggregating platelets cause contraction of vascular smooth muscle, because they release serotonin and thromboxane A2. If the platelets aggregate in a blood vessel with intact intima, the platelet-products cause endothelium-dependent relaxation of the underlying smooth muscle. Hence, the presence of an intact intima considerably reduces the vasospastic response to platelet-aggregation. The major platelet products which trigger endothelium-dependent relaxations are the adenine nucleotides and serotonin. The ability of the endothelium to prevent platelet-induced vasospasm is augmented after chronic intake of cod liver oil, but is reduced after previous intimal injury.     

A rapid HPLC method for determination of adenylate energy charge

A simple procedure is described for separation and analysis of adenine nucleotides in tissue extracts, utilizing anion exchange HPLC. Determination of AMP, ADP, and ATP takes 10 min per sample.     

Topographical distribution of ATP in rat brain.

Studies on the distribution of ATP in microdissected segments of the rat brain indicate that the nucleotide is concentrated in gray matter, and especially in the thalamus, hippocampus, entorhinal cortex and sensorimotor cortex. These distribution studies in conjunction with previous neuropharmacological studies, support the concept that adenine nucleotides may function as intercellular mediators in various regions of the brain.     

An assessment of the effect of hormone and neurotransmitters on adenine and guanine derivatives simultaneously in rat brain cortical slices.

A A 2-dimensional thin-layer method has been developed for the separation on cellulose of adenine and guanine derivatives. Using incubated rat cerebral cortex slices it was shown that noradrenaline and acetylcholine stimulated cAMP and cGMP production respectively but glutamate and gamma-aminobutyric acid stimulated production of both cyclic nucleotides.     

Topographical distribution of ATP in rat brain

Summary Studies on the distribution of ATP in microdissected segments of the rat brain indicate that the nucleotide is concentrated in gray matter, and especially in the thalamus, hypothalamus, hippocampus, entorhinal cortex and sensorimotor cortex. These distribution studies in conjuction with previous neuropharmacological studies, support the concept that adenine nucleotides may function as intercellular mediators in various regions of the brain.     

A comparison of the effects of the optical isomers of isoproterenol on energy metabolism in a mouse sarcoma

Summary Disturbance to energy production in the S180 sarcoma (CB) by optical isomers of isoproterenol was assessed from altered adenine nucleotide levels at 1 h. The L-isomer almost halved the ATP level and lowered the energy charge significantly; the D-isomer was inactive. Dependence of tumor injury on cytochrome P-450 activity appears unlikely.One of us (GRNJ) thanks the Department of Surgery, Medical School, Kings's College Hospital, London, England, for the provision of experimental facilities; the Institute of Biochemistry, German Cancer Research Centre, Heidelberg, FRG, for permitting the measurement of metabolites; the Cancer Research Campaign, London, UK, for a part-time grant; and Zyma GmbH, München, FRG, and the estate of the late Dr Lucie Polak for additional financial support.     

An assessment of the effect of hormone and neurotransmitters on adenine and gluanine derivatives simultaneously in rat brain cortical slices

Summary A 2-dimensional thin-layer method has been developed for the separation on cellulose of adenine and guanine derivatives. Using incubated rat cerebral cortex slices it was shown that noradrenaline and acetylcholine stirnulated cAMP and cGMP production respectively but glutamate and -aminobutyric acid stimulated production of both cyclic nucleotides.     

Myocardial force production and energy turnover in anoxia

ATP turnover of isolated rabbit papillary muscles, contracting isometrically at 20 degrees C, was determined in oxygen and during 40 min of exposure to nitrogen (anoxia). Stimulus frequency was 0.2 hertz (Hz) in oxygen and 0.2 or 1.0 Hz in nitrogen. In oxygen, ATP turnover was determined from oxygen consumption using a P/O2 ratio of 6.3. The time-dependent rate of ATP turnover in nitrogen was found from the production of lactate, and the changes in adenine nucleotides and phosphocreatine, measured in rapidly frozen preparations at different time-points during the anoxic period. A P/lactate ratio of 1.5 was used. In muscles stimulated at 0.2 Hz, twitch force dropped during the anoxic period to 33% while force production of muscles stimulated at 1.0 Hz stopped completely. However, in the latter muscles, resting force rose to 19% of the twitch force in oxygen. The rate of ATP hydrolysis in anoxia depended strongly on stimulus frequency, indicating that it is not solely determined by the glycolytic capacity. In the 0.2 Hz-stimulated muscles the decrease in energy turnover occurred in parallel with the drop in force. However, the rise in resting force in muscles stimulated at 1.0 Hz occurred when ATP turnover was close to zero. It was concluded that anoxia hardly affects the energy required for twitch force production, but that the rise of resting force measured when twitch force had disappeared occurred when the rates of cross-bridge cycling and calcium turnover were very low.     

Die oxydative Phosphorylierung isolierter Mitochondrien und ihre Abhängigkeit von strukturgebundenen Faktoren

Summary The ability of isolated mitochondria in coupling phosphorylation to oxidation depends on sufficient concentrations of intramitochondrially bound adenine nucleotides (especially ATP), compounds of oxidative metabolism (pyruvic and lactic acid) and iron ions. Continuous loss of these components by ageing causes at first a parallel lowering of phosphate uptake and does not affect respiration. Later on, a decrease of oxidative ability begins when the loss of intramitochondrial components reaches a limiting concentration, and now mitochondria show more and more swelling and structural damages.      

Inhibition of rat liver transglutaminase by nucleotides

Summary Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Inhibition of rat liver transglutaminase by nucleotides.

Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Bestimmung von Glykolyse-Metaboliten in isolierten Zellkernen

Summary Isolated rat liver nuclei have been analyzed for the intranuclear concentration of 8 glycolytic metabolites, and in addition for the 3 adenine nucleotides, and for malate. All these substrates are to be found in nuclei in a range of concentration comparable to that in the whole tissue. The consequences of these findings with respect to the definite establishment of glycolysis in isolated nuclei, as well as for the intranuclear formation of energy-rich phosphate bonds, are discussed.

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Meinem Lehrer, Prof. Dr. Dr.K. Lang, in aller Verehrung und Dankbarkeit zum 60. Geburtstag gewidmet.     

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O₂ consumption and H₂O₂ release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H₂O₂, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H₂O₂ release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation. Received 18 May 2001; received after revision 17 July 2001; accepted 24 July 2001     

电动汽车锂离子动力电池充放电性能试验分析

根据电动汽车动力电池研发需求,对锂离子单体电池进行了一系列充放电试验,得到了该电池在不同放电倍率,以及不同温度条件下的充放电特性、开路电压、温升、内阻与效率特性.结果表明,锂离子电池具有比能量高、内阻小、放电效率高、放电特性良好等优点;锂电池的荷电状态与电池开路电压存在近似线性关系,这使得利用开路电压结合安时法估计电池的SOC成为现实.     

Modulation of hepatic mitochondrial energy efficiency with age

This study was designed to examine the effect of youth-adulthood transition on hepatic mitochondrial energy efficiency. The changes in basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, were evaluated in mitochondria isolated from the liver of young and adult rats. Alterations in mitochondrial cytochrome oxidase and aconitase specific activities, and in adenine nucleotide translocator content were also assessed. There was no difference in basal proton leak or thermodynamic coupling and efficiency of oxidative phosphorylation in liver mitochondria between the two rat groups. On the other hand, palmitate-induced proton leak increased significantly in adult rats. The function of this uncoupling could be avoidance of elevated formation of reactive oxygen species, which are known to accelerate ageing.Received 17 February 2004; received after revision 30 March 2004; accepted 1 April 2004     

Circadian variation of adrenocortical cyclic nucleotides (cyclic AMP and cyclic GMP) in hypophysectomized rats

The existence of a circadian variation in the adrenocortical concentrations of cyclic AMP and cyclic GMP in male adult Wistar rats examined 10 days after hypophysectomy is demonstrated. The results suggest that the circadian variations of adrenocortical cyclic nucleotides observed previously in intact rats might not entirely depend upon pituitary corticotrophin.