Oxidative stress and adenine nucleotide control of mitochondrial permeability transition

Mitochondria can initiate apoptosis by releasing cytochrome c after undergoing a calcium-dependent permeability transition (MPT). Although the MPT is enhanced by oxidative stress and prevented by adenine nucleotides such as adenosine 5'-diphosphate (ADP), the hypothesis has not been tested that oxidants regulate the effects of exogenous adenine nucleotides on the MPT and cytochrome c release. We found that cytochrome c release from intact rat liver mitochondria depended strictly on pore opening and not on membrane potential, and that MPT-enhancing oxidative stress also augmented cytochrome c release. At low oxidative stress, micromolar (ADP) and low adenosine 5'-triphosphate (ATP)/ADP ratio inhibited the MPT and cytochrome c release, whereas ATP or high ATP/ADP had only a slight effect. In freshly isolated mitochondria, the time to half-maximal MPT was related to the log of the ATP/ADP ratio. This function was shifted to shorter times by oxidative stress which decreased ADP protection and caused ATP to accelerate the calcium-dependent MPT. By comparison, mitochondria treated with reducing agents and those isolated from septic rats were protected from the MPT by both nucleotides. These results indicate that oxidation-sensitive site(s) in the membrane regulate the effects of adenine nucleotides on the MPT. The oxidant-based differences in the effects of ADP and ATP on the pore support the novel hypothesis that failure of the cell to consume ATP and provide adequate ADP at the adenine nucleotide transporter during oxidative stress predisposes to cytochrome c release and initiation of apoptosis.     

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum

Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Inhibition by adenine compounds of the induced formation of photosynthetic pigments in Rhodopseudomonas spheroides cells under dark-semiaerobic conditions

Effects of adenine, adenosine, AMP, ADP and ATP on the inducedformation of bacteriochlorophyll and carotenoids in cell suspensionsof dark-aerobically grown Rhodopseudomonas spheroides were examinedunder dark-semiaerobic conditions where no significant cellgrowth occurred. Pigment formation was strongly inhibited by3 mM adenine, adenosine, AMP or ATP, but less strongly by ADP.Inhibition by either adenosine or ATP was completely reversible.Addition of 3 mM adenosine resulted in complete inhibition ofpigment formation, while inhibition by more than 10 min ATPdid not exceed 80%. No accumulation of any precursor-like pigmentsof either bacteriochlorophyll or carotenoids was observed incells incubated in the presence of adenine compounds. Amountsof exogenously-added adenine, adenosine, or AMP decreased significantlyduring incubation, whereas the amount of exogeneously-addedATP or ADP did not appreciably decrease. Addition of 3 mM ATPor adenosine also significantly suppressed ³H-leucine incorporationinto bacterial proteins. Nucleosides other than adenosine wereineffective in inhibiting the induced formation of photosyntheticpigments, indicating that the inhibitory action is specificto adenine compounds. It was assumed that both adenosine andATP inhibit chromatophore formation rather than a particularstep(s) in the biosynthetic pathways of the photosynthetic pigment,and that ATP exerts its effect from outside the cells, whereasadenosine does so after being taken up by the cells. (Received July 24, 1972; )     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Compartmentalized ATP pools produced from adenosine are nuclear pools

Incubation of African green monkey kidney (BS-C-1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5'-triphosphate (ATP) and nuclear ATP/adenosine 5'-diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine-promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS-C-1 cells than in the spontaneously transformed 3T6 cells. Adenosine-promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all of the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5'-monophosphate (AMP) deaminase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively--conditions which were shown by earlier in vitro studies to be favorable to DNA replication.     

The purine nucleotide cycle. Studies of ammonia production and interconversions of adenine and hypoxanthine nucleotides and nucleosides by rat brain in situ.

Electrical shock treatment produces a rapid loss of high energy phosphates in rat brain. The [ATP]/[ADP] ratio decreases to one-third of its control value within 10 s. The ammonia content increases 3-fold during the first minute after starting the stimulus. The total adenine nucleotide plus adenosine content of brain decreases an equivalent amount of hypoxanthine-containing compounds appears. Adenosine, inosine, and hypoxanthine accumulate, and there is a transitory accumulation of adenylosuccinate. The contents of ATP and creatine phosphate, and the [ATP]/[ADP] ratio, are rapidly restored to control values, but other metabolite contents are restored more slowly. The transient rise in adenylosuccinate and IMP provides evidence that the ammonia production is due in part, and possibly in whole, to the operation of the purine nucleotide cycle.     

Evidence for two adenine derivative receptors in rat ileum which are not involved in the nonadrenergic, noncholinergic response.

The receptors mediating inhibition of the rat ileum by adenosine and adenine nucleotides were studied. ATP and ADP were more potent than AMP or adeonsine. Theophylline antagonized the effects of adenosine and AMP but not those of ATP or ADP. Preparations desensitized to ATP or ADP were still inhibited by adenosine and vice versa. The nonadrenergic, noncholinergic inhibition produced by field stimulation or nicotine was not attenuated by the presence of theophylline or desensitization to ATP. These data indicate that more than one adenine derivative receptor is present in rat ileum and that ATP and adenosine are unlikely candidates for the unknown transmitter.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes

Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7-8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine.     

Extracellular adenine compounds, red blood cells and haemostasis: facts and hypotheses

Previously, the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. Research of the last decades has revealed that nucleotides also occur in the extracellular milieu. This survey deals with extracellular adenine compounds in the blood, focussing on their role as chemical mediators in the haemostatic effect of red cells. Erythrocytes may act as pro-aggregatory cells by at least two chemical mechanisms. Firstly, they can enhance platelet aggregation by releasing adenosine diphosphate (ADP), a well known platelet stimulatory substance. ADP is set free when red cells are stressed mechanically, for instance by shear forces generated in the blood stream; ample experimental evidence supporting this view is summarized. Secondly, erythrocytes efficiently take up extracellular adenosine via their nucleoside transporters, thereby removing a potent inhibitor of platelet function. Extracellular adenosine occurs in the blood stream, either directly released from various tissues or as the end product of extracellular adenine nucleotide metabolism, e.g. after degradation of red cell-born ADP or ATP. Finally, a novel mechanism of action of the antithrombotic drug dipyridamole, which has very recently been put forward, is demonstrated. Dipyridamole inhibits platelet function indirectly by blocking the uptake of extracellular adenosine via the nucleoside transporter of red cells; increased adenosine levels in turn are responsible for the antiaggregatory effect of dipyridamole.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Effects of hypothermia on canine kidney mitochondria

The effect of hypothermia on the function of isolated dog kidney cortex mitochondria was determined with an FAD- and NAD⁺-linked substrate. In dog kidney mitochondria, temperatures of 10 °C or less suppress ADP stimulation of respiration but have little or no effect upon uncoupler, Ca²⁺ or valinomycin-K⁺ stimulation of respiration. This suggests that the adenine nucleotide translocase which catalyses the transport of ADP into the mitochondria limits the rate of respiration and generation of ATP at 10 °C in kidneys undergoing preservation. The coupling of oxidation to phosphylation, as determined by measuring the amount of ATP formed at low temperatures, indicates, however, that mitochondria are fully coupled at both 10 and 5 °C. The respiratory control index at 15 °C is greater (with pyruvate plus malate) than at 30 or 10 °C and suggests that 15 °C may be the optimum perfusion temperature for maintaining adenine nucleotide levels in the perfused kidney.     

In vitro effect of homocysteine on nucleotide hydrolysis by blood serum from adult rats

During the past few years, elevated blood levels of homocysteine (Hcy) have been linked to increased risk of premature coronary artery disease, stroke and thromboembolism. These processes can be also related to the ratio adenine nucleotide/adenosine, since extracellularly these nucleotides are associated with modulation of processes such as platelet aggregation, vasodilatation and coronary flow. Furthermore, there are some studies that suggest a relationship between Hcy and plasma adenosine concentrations. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes one of the systems for rapid inactivation of circulating adenine nucleotides. Thus, the main objective of this study was to evaluate if Hcy can participate in the modulation of the extracellular adenine nucleotide hydrolysis by rat blood serum. Our results showed that Hcy, at final concentrations of 5.0 mM, inhibits in vitro ATP, ADP and AMP hydrolysis by 26, 21 and 16%, respectively. Also Hcy, at final concentrations of 8.0mM, inhibited the in vitro hydrolysis of ATP, ADP and AMP by 46, 44 and 44%, respectively. Kinetic analysis showed that the inhibitions of the three adenine nucleotide hydrolyses in the presence of Hcy, by serum of adult rats, is of the uncompetitive type. The IC50 calculated from the results obtained were 6.52+/-1.75 mM (n = 4), 5.18 +/- 0.64 mM (n = 3) and 5.16 +/- 1.22 mM (n = 3) for ATP, ADP and AMP hydrolysis, respectively.     

Adenosine Receptor Activation and the Regulation of Tyrosine Hydroxylase Activity in PC 12 and PC 18 Cells

We compared the response of rat PC12 cells and a derivative PC18 cell line to the effects of adenosine receptor agonists, antagonists, and adenine nucleotide metabolizing enzymes. We found that theophylline (an adenosine receptor antagonist), adenosine deaminase, and AMP deaminase all decreased basal cyclic AMP content and tyrosine hydroxylase activity in the PC12 cells, but not in PC18 cells. Both cell lines responded to the addition of 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine, adenosine receptor agonists, by exhibiting an increase in tyrosine hydroxylase activity and cyclic AMP content. The latter finding indicates that both cell lines contained an adenosine receptor linked to adenylate cyclase. We found that the addition of dipyridamole, an inhibitor of adenosine uptake, produced an elevation of cyclic AMP and tyrosine hydroxylase activity in both cell lines. Deoxycoformycin, an inhibitor of adenosine deaminase, failed to alter the levels of cyclic AMP or tyrosine hydroxylase activity. This suggests that uptake was the primary inactivating mechanism of adenosine action in these cells. We conclude that both cell types generated adenine nucleotides which activate the adenosine receptor in an autocrine or paracrine fashion. We found that PC12 cells released ATP in a calcium-dependent process in response to activation of the nicotinic receptor. We also measured the rates of degradation of exogenous ATP, ADP, and AMP by PC12 cells. We found that the rates of metabolism of the former two were at least an order of magnitude greater than that of AMP. Any released ATP would be rapidly metabolized to AMP and then more slowly degraded to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of adenine nucleotides in thymocyte cytoplasm and subcellular fractions after treatment with concanavalin A, papaverine and adenosine

Studies with rat thymocytes labeled with [14C]adenine and fractionated by digitonin treatment revealed that the cytoplasm of these cells contains about 60% of the total adenine nucleotide pool with a higher ATP/ADP ratio and metabolic activity as compared with the structural components. The incorporation of [14C]adenine and [14C]adenosine into thymocyte adenine nucleotides results in predominant labeling of cytoplasmic ATP, in which the specific radioactivity of this nucleoside triphosphate is two and three times as high as in subcellular structures. Concanavalin A decreases the ATP level in thymocytes without changing its specific radioactivity. This compound does not influence the total content and amount labeled adenine nucleotides in the structural fraction. Papaverine accelerates the catabolism of ATP, mainly in thymocyte cytoplasm and, in a lesser degree, in its structural fraction. In each fraction the papaverine-induced catabolism of ATP is localized in the compartment which is more intensively labeled with [14C]adenine than the whole fractionation ATP pool. Adenosine markedly accelerates adenine nucleotide catabolism in the cytoplasmic and structural fractions of thymocytes; however, only in the first one of them this acceleration is due to ATP elevation. Papaverine and adenosine do not directly influence either the content or specific radioactivity of adenine nucleotides of the structural fraction isolated from [14C]adenine-labeled thymocytes.     

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.     

Fluorometric determination of adenine nucleotides and adenosine by ion-paired, reverse-phase, high-performance liquid chromatography

A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).