Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N⁶-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Purification of cofactor-dependent enzymes by affinity chromatography

Several 8-(6-aminohexyl)-amino adenine nucleotide derivatives, including ATP, 2′,5′-ADP, 3′,5′-ADP and desulfo-CoA (CoA, reduced coenzyme A), were prepared and immobilized on Sepharose by cyanogen bromide activation. 8-(6-Aminohexyl)-amino-ATP-Sepharose was found to exhibit good affinity for both NAD⁺-dependent dehydrogenases and kinases. Sequential biospecific elutions with NADH and ATP resulted in a good separation of dehydrogenases from kinases. As many as eight different dehydrogenases and kinases could be substantially purified from both porcine muscle and mouse kidney extracts by this new procedure. 8-(6-Aminohexyl)-amino-2′,5′-ADP- and −3′,5′-ADP-Sepharose were shown to exhibit good affinity for many NADP⁺-dependent dehydrogenases from yeast extracts and CoA-dependent enzymes, respectively. Purification of citrate synthases from pig heart and Eschericia coli extracts by means of these 8-substituted adenine nucleotide affinity columns was also presented.     

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)     

Biosynthesis of cytokinins

Cytokinins are adenine derivatives with an isoprenoid side chain and play an essential role in plant development. Plant isopentenyltransferases that catalyze the first and rate-limiting steps of cytokinin biosynthesis have recently been identified. Unlike bacterial enzymes, which catalyze the transfer of the isopentenyl moiety from dimethylallyldiphosphate (DMAPP) to the N ⁶ position of adenosine 5′-monophosphate (AMP), plant enzymes catalyze the transfer of the isopentenyl moiety from DMAPP preferentially to ATP and to ADP. The isopentenylated side chain is hydroxylated to form zeatin-type cytokinins. An alternative pathway, in which a hydroxylated side chain is directly added to the N ⁶ position of the adenine moiety, has also been suggested.     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.     

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; )     

The reaction of 14C-labelled platinum ethylenediamine dichloride with adenine compounds and DNA

Various derivatives of adenine have been studied with regard to their rate of reaction with ¹⁴C-labelled platinum ethylenediamine dichloride, Pt(¹⁴C-en)Cl₂. The reactivities have been calculated from the “rate of disappearance” of Pt(¹⁴C-en)Cl₂ using chromatographic separation of reactants and products.Adenine and adenosine react very slowly at 37° whereas other adenine derivatives react much more readily in the order: poly A > AMP > ApA > poly d(AT). From the numerical values of the rate constants it is concluded that the presence of a phosphate group increases the reaction rate considerably. This is partly the explanation of the rapid reaction of poly A which possesses terminal phosphate groups. However adjacent adenine moieties such as those in polyadenylic acid (poly A) and adenosyl-3′5′-adenosine (ApA) may also react by another mechanism which involves the 6-NH₂ groups.The energies of activation of the second order reaction with platinum ethylenediamine dichloride (PtenCl₂) are 12.9, 18.8, 19.0 kcal/mole for poly A, AMP and ApA respectively.In DNA, no free phosphate groups are present, and the occurrence of adjacent adenines will be low. The reaction of PtenCl₂ with DNA seems to involve a rapid attack on deoxyguanosine (GdR) and a slow reaction with deoxyadenosine (AdR) and deoxycytidine (CdR).     

ATP formation from deoxyadenosine in human erythrocytes: Evidence for a hitherto unidentified route involving adenine and S-adenosylhomocysteine hydrolase

A novel route of ATP formation has been identified using erythrocytes from patients deficient in four different enzymes associated with ATP formation. It entails prior adenine production from deoxyadenosine (or adenosine) in a reaction involving S-adenosylhomocysteine hydrolase. The postulated route has been demonstrated in human erythrocytes which, unlike other human cells, cannot form ATP from IMP. It is based on studies by others using purified S-adenosylhomocysteine hydrolase preparationsin vitro. The results provide the first confirmation that this reaction occurs in intact human cellsin vitro and thus most probablyin vivo. This adenine production is normally masked in intact cells by further metabolism to ATP. Clinical significance for such a route is suggested by the fact that some adenosine analogues with potent oncostatic and antiviral properties also release adenine (or analogues)in vitro.     

Effect of various adenine derivatives on gastric acid secretion in the isolated rat stomach

The adenine derivatives adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine (AD), in concentrations of 10(-3)M and 10(-4)M caused significant and dose-related modifications in the basal acid secretion from isolated whole rat stomach. The first three purine derivatives, ATP, ADP and AMP significantly increased the spontaneous acid secretion. On the other hand, AD caused a significant reduction in the basal acid secretion. When ATP, ADP, AMP and AD were assayed in the presence of the adrenergic and cholinergic blocking agents, ergotamine, 10(-6)M, propranolol, 5 X 10(-7)M and atropine, 10(-6)M, all these purine derivatives, including AD, caused a significant increase in the basal acid secretion.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

The metabolism of adenine derivatives in different parts of the brain of the rat,and their release from hypothalamic preparations on excitation

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [¹⁴C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [¹⁴C]adenine, the hypothalamic tissues released about 0.2% of their ¹⁴C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 μUM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned, and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

A new chromatographic method using immobilized acriflavin for measuring cyclic AMP in cells prelabeled with radioactive adenine.

A method using the principle of charge-transfer chromatography has been developed for the determination of cyclic AMP levels in intact prelabeled cells. The ATP pool was prelabeled by incubating the cells in the presence of radioactive adenine. The cyclic AMP formed from ATP was extracted with HC10(4) and separated from adenine and other adenosine-related nucleotides by chromatography on acriflavin-Sephadex G-25. This method provides a rapid and sensitive isolation of cyclic AMP with high recovery (95-100%) and low blnks. Further, no contamination of the cyclic AMP fractions was found by either adenine or adenosine nucleotides such as ATP, ADP or AMP. This procedure is applicable to a variety of cell or tissue systems.     

5'-adenine mononucleotides in preparations from guinea pig cerebral cortex. Accumulation, translocation and release.

Isolated nerve terminals (synaptosome beds) were prepared from the neocortex of guinea pig and their ability to accumulate and release adenine nucleotides was studied. Synaptosome beds prelabelled with [14C]adenosine released newly synthesized [14C] adenine derivatives on superfusion. Electrical stimulation and K+ depolarization gave augmented output of both [14C] adenine derivatives and lactate from the preparations. Action of metabolic inhibitors on this output was examined. During incubation and superfusion, the synaptosomes displayed glycolysis and synthesis of ATP. Supply of adenine derivatives to the nerve terminals also occurred by translocation from other parts of the tissue.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Inhibition of a calcium-activated, non-selective cation channel, in a rat insulinoma cell line, by adenine derivatives

The effects of adenosine and adenine nucleotides on a calcium-activated non-selective cation channel, present in the plasma membrane of an insulin-secreting cell line CRI-Gl were investigated. Single-channel currents were recorded from inside-out membrane patches and the adenine derivatives applied to the solution bathing the cytoplasmic aspect of the membrane surface. The activity of this channel is shown to be inhibited by all the derivatives tested. The potency sequence for inhibition was found to be AMP greater than ADP greater than ATP greater than adenosine.     

ATP and related purines stimulate motility,spatial congregation,and coalescence in red algal spores

Adenosine 5′‐triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration‐response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time‐dependent manner. 10–300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2–2.5 μm · s^?1. 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7‐fold as compared with parallel controls. Likewise, 1–10 μM cAMP or 30–100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.