A rapid method for measuring ATP + ADP + AMP in marine sediment

In this report, I describe a method for rapid measurement of total adenylate (ATP + ADP + AMP) in marine sediment samples for estimating microbial biomass. A simple ‘boil and dilute’ method is described here, whereby adding boiled MilliQ water to sediments increases the detection limit for ATP + ADP + AMP up to 100-fold. The lowered detection limit of this method enabled the detection ATP + ADP + AMP in relatively low-biomass sub-seafloor sediment cores with 10⁴ 16S rRNA gene copies per gram. Concentrations of ATP + ADP + AMP correlated with 16S rRNA gene concentrations from bacteria and archaea across six different sites that range in water depth from 1 to 6000 m indicating that the ATP + ADP + AMP method can be used as an additional biomass proxy. In deep sea microbial communities, the ratio of ATP + ADP + AMP concentrations to 16S rRNA genes >1 m below seafloor was significantly lower compared to communities in the upper 30 cm of sediment, which may be due to reduced cell sizes and or lower ATP + ADP + AMP concentrations per cell in the deep sea sub-seafloor biosphere. The boil and dilute method for ATP + ADP + AMP is demonstrated here to have a detection limit sufficient for measuring low biomass communities from deep sea sub-seafloor cores. The method can be applied to frozen samples, enabling measurements of ATP + ADP + AMP from frozen sediment cores stored in core repositories from past and future international drilling campaigns.     

Evaluation of the BOD POD for estimating percent fat in female college athletes

The purpose of this investigation was to examine the accuracy of percent body fat (%BF) estimates obtained by air displacement plethysmography (ADP) using the BOD POD Body Composition System compared with hydrostatic weighing (HW) in a group of female college athletes (n = 80). In addition, %BF estimates by skinfold measures (SF) were also obtained for comparison. A lean subset (n = 39) of the sample was also examined. Mean %BF estimated for the entire sample by ADP (21.2 +/- 5.9%) was significantly greater than that determined by HW (19.4 +/- 6.4%) and SF (18.8 +/- 5.5%). Results from the lean subset also revealed that %BF determined by ADP (17.1 +/- 3.7%) was significantly higher than %BF estimates by HW (14.3 +/- 2.8%) and SF (15.2 +/- 3.2%). The regression equation for the entire sample (%BF HW = 0.937%BF ADP - 0.452, r(2) = 0.73, standard error of estimates (SEE) = 3.34) did not differ from the line of identity. In contrast, the line of identity differed significantly from the regression equation for the lean subset of female athletes (%BF HW = 0.48%BF ADP + 6.115, r(2) = 0.41, SEE = 2.18). The results of this investigation indicate that ADP significantly overestimated %BF by 8% in female athletes and by 16% for a leaner subset of the sample compared with HW. It appears that %BF estimates by SF may be more accurate than those obtained by ADP for female college athletes, regardless of body composition. Coaches and trainers evaluating body composition should consider the use of SF before ADP when measuring %BF in female college athletes. Sports scientists should continue to examine the possible gender and body composition bias for ADP.     

Effects of covert subject actions on percent body fat by air-displacement plethsymography

Air-displacement plethysmography (ADP) is used for estimation of body composition, however, some individuals, such as athletes in weight classification sports, may use covert methods during ADP testing to alter their apparent percent body fat. The purpose of this study was to examine the effect of covert subject actions on percent body fat measured by ADP. Subjects underwent body composition analysis in the Bod Pod following the standard procedure using the manufacturer's guidelines. The subjects then underwent 8 more measurements while performing the following intentional manipulations: 4 breathing patterns altering lung volume, foot movement to disrupt air, hand cupping to trap air, and heat and cold exposure before entering the chamber. Increasing and decreasing lung volume during thoracic volume measurement and during body density measurement altered the percent body fat assessment (p < 0.001). High lung volume during thoracic gas measures overestimated fat by 3.7 ± 2.1 percentage points. Lowered lung volume during body volume measures overestimated body fat by an additional 2.2 ± 2.1 percentage points. The heat and cold exposure, tapping, and cupping treatments provided similar estimates of percent body fat when compared with the standard condition. These results demonstrate the subjects were able to covertly change their estimated ADP body composition value by altering breathing when compared with the standard condition. We recommend that sports conditioning coaches, athletic trainers, and technicians administering ADP should be aware of the potential effects of these covert actions. The individual responsible for administering ADP should remain vigilant during testing to detect deliberate altered breathing patterns by athletes in an effort to gain a competitive advantage by manipulating their body composition assessment.     

Extracellular metabolisation of NADH by blood cells correlates with intracellular ATP levels

A new assay allowing quantitation of extracellular NADH metabolisation by intact blood cells was compared with the intracellular ATP/ADP ratio of these cells. The sensitivity, reproducibility and NADH specificity of this assay were determined. The diagnostic potential of this test was examined in a study with highly conditioned athletes. NADH consumption was measured before and immediately after maximum aerobic performance as well as 1 day later and was compared with the ATP/ADP level in these blood cells. A significant decline of cellular energy after aerobic performance was detected with both approaches to a similar extent (P<0.01). However, the extracellular NADH metabolisation assay (ENMA) is more convenient to perform than the determination of intracellular ATP/ADP. Due to its easy and versatile handling, a huge array of possible applications like monitoring the training efficiency of athletes, the fitness of senior citizens or the recovery from disease may be envisioned.     

Characterization of the NTPDase activities in the mesentery pre- and post-capillary circuits of the guinea pig

NTPDase is one of the principal enzymes involved in the sequential hydrolysis of ATP. In the present study, the presence and functionality of NTPDase in the mesenteric vein and artery were examined. Adenosine triphosphate (ATP) (0.01-1000 pmol) induces a dose-dependent vasodilation in the isolated arterial and venous mesenteric vasculatures of the guinea pig. Adenosine diphosphate (ADP) (0.01-1000 pmol) but not adenosine monophosphate (AMP) (0.01-1000 pmol) induces a similar response in the mesenteric vascular circuit. L-NAME, a nitric oxide synthase inhibitor (200 microM, 30 min), significantly reduces the arterial dilatory effect of ATP and abolishes the responses to ADP and AMP. Complete removal of the endothelium with 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate (CHAPS) (20 mM, 2 x 45 s) abolishes ATP-induced responses. Infusion of ATP in the vascular circuit generated detectable amounts of ADP and AMP, as measured by HPLC. CHAPS treatment significantly reduced the level of ATP and the production of AMP in the arterial mesenteric circuit. In contrast to the arterial mesenteric vasculature, endothelium removal in the venous circuit triggered a marked potentiation of ADP release and, interestingly, a marked reduction in the release of AMP. Moreover, a specific inhibitor of NTP diphosphohydrolase, 1-hydroxynaphthlene-3,6-disulfonic acid BGO 136 (10 mM for 20 min), significatively reduced AMP production in both vascular preparations. These results confirm that the endothelium contributes to the vasoactive properties of ATP, ADP, and AMP. Our data also demonstrated a significant role of endothelium in NTPDase activity on ADP and AMP production prior to exogenous administration of ATP. The activity of this particular enzyme appears to be different from the reaction products viewpoint (i.e., the production of ADP) in the pre- and post-mesenteric circuits, suggesting two different isoforms with different substrate specificities.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

Synthesis of bound adenosine triphosphate from bound adenosine diphosphate by the purified coupling factor 1 of chloroplasts. Evidence for direct involvement of the coupling factor in this "adenylate kinase-like" reaction.

Electrophoretically homogeneous coupling factor 1 from spinach chloroplasts binds ADP and converts the bound ADP to bound ATP and AMP. That this transphosphorylation of enzyme-bound ADP is catalyzed by the coupling factor itself, and not be a conventional adenylate kinase which might possibly contaminate preparations of the coupling factor, is supported by the following evidence. 1. The procedure for isolatio of the coupling factor is designed to separate this large (approximately 13 S) enzyme from the smaller (4.2 S) conventional adenylate kinase of spinach chloroplasts. The conventional adenylate kinase cannot be detected in purified preparations of the coupling factor by biochemical assay or by polyacrylamide gel electrophoresis. 2. The activity of spinach adenylate kinase is completely dependent upon magnesium ions. However, the production of bound ATP and AMP from bound ADP by the coupling factor can be assayed in the total absence of added magnesium ions or even in the presence of added EDTA. 3. Comparative studies with inhibitors show that the coupling factor can produce bound ATP from ADP under conditions where the activity of adenylate kinase is strongly inhibited. Conversely, the coupling factor is prevented from synthesizing bound ATP from ADP under other conditions where the conventional adenylate kinase has high levels of activity. 4. AMP, when added in solution to the coupling factor, does not bind to this enzyme, even in the presence of APT. Thus, it is unlikely that the appearance of AMP bound to the coupling factor after its incubation with ADP is due to the production of free AMP by contaminating adenylate kinase. These results demonstrate that the isolated, homogeneous coupling factor from spinach chloroplasts has the intrinsic capacity to perform a phosphoryl group transfer between two bound ADP molecules and thus to synthesize ATP. This reaction may have an important role in the photosynthetic production of ATP by the chloroplast, as is discussed in this communication.     

AMP photophosphorylation and binding by chloroplasts

Stoichiometric amounts of chloroplast thylakoids photophosphorylate free AMP to tightly bound ADP. Free ADP is a poor competitor for this AMP photoreaction, which saturates below 16 micronAMP. The inhibitor, diadenosine pentaphosphate, abolishes AMP photophosphorylation, and inhibits dark ADP binding. Taken together, these data imply that this photoreaction involves the high affinity nucleotide binding site(s) of chloroplast coupling factor CF1, and that little mixing with free nucleotides occurs.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Structural insight into AMPK regulation: ADP comes into play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.     

Adenosine kinase from rat liver: new biochemical properties

Adenosine kinase is a well-known enzyme which catalyzes the phosphorylation of adenosine to AMP: Its metabolic and kinetic properties are well studied. Here, we report new properties of rat liver enzyme, demonstrating a new reaction: ADP can be a phosphate donor instead ATP, according to the reaction: adenosine + ADP --> 2AMP) demonstrating the efficiency of AdK to phosphorylate adenosine, also starting from ADP. Cells could exploited this property in situations in which ATP levels are strongly decreased and ADP decreases slowly.     

Adenosine Kinase from Rat Liver: New Biochemical Properties

Adenosine kinase is a well-known enzyme which catalyzes the phosphorylation of adenosine to AMP: Its metabolic and kinetic properties are well studied. Here, we report new properties of rat liver enzyme, demonstrating a new reaction: ADP can be a phosphate donor instead ATP, according to the reaction: adenosine + ADP → 2AMP) demonstrating the efficiency of AdK to phosphorylate adenosine, also starting from ADP. Cells could exploited this property in situations in which ATP levels are strongly decreased and ADP decreases slowly.     

The effect of adenine nucleotides and inhibitors of ADP translocation on uncoupled electron flux in bean mitochondria

The uncoupled electron flux and the influence of adenine nucleotides on this flux in mitochondria isolated from hypocotyls of Vigna sinensis (L.) Savi cv. Seridó were examined. In order to avoid the functioning of other enzymes capable of utilizing adenine nucleotides the reaction medium was free of Mg²⁺. When an oxidizable NADH -linked substrate such as L-malate was used, a stimulatory of adenosine-5′-monophosphate (AMP) and adenosine-5′-diphosphate (ADP) on uncoupled respiration was manifested. The stimulatory effect of AMP and ADP could not be shown when succinate was the substrate. Atractyloside and carboxyatractyloside had no effect on the stimulatory role played respectively by AMP and ADP in the presence of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP).     

The structure of the nucleotide-binding site of kinesin.

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.     

Adenylate kinase as a virulence factor of Pseudomonas aeruginosa

Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg. ATP + AMP <--> Mg. ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.     

Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea-pig hearts. Whole-tissue adenylate data from freeze-clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non-aqueous media. Adenosine and inosine in venous cardiac effluents were measured by high-performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non-aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near-quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were predominantly located in the cytosol of the well-oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP [( CrP]/[Cr] X [Pi]) and hence that of ATP [( ATP]/[ADP] X [Pi]) assuming the creatine kinase at near-equilibrium at a near-constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearly up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass-action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher-power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearly correlated (r = 0.920) with the calculated free cytosolic AMP concentration. Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Airway effects of purine nucleosides and nucleotides and release with bronchial provocation in asthma

Adenosine, AMP, and ADP all caused similar concentration-related bronchoconstriction when inhaled by patients with asthma, whereas the adenosine hydrolysis product inosine had no effect. Geometric mean provocation concentrations of adenosine AMP and ADP causing a 20% fall in forced expiratory volume in 1 s (PCf20) were 2.34, 4.27, and 2.19 mumol/ml and 40% fall in specific airway conductance (PCs40) 3.16, 5.01, and 2.0 mumol/ml. Bronchoconstriction was rapid in onset, reaching a maximum 2-5 min after a single inhalation of AMP. In 31 asthmatic subjects a positive correlation was established between airway responsiveness to histamine, as an index of non-specific responsiveness, and airway reactivity to adenosine (PCf20, r = 0.60; PCs40, r = 0.64; P less than 0.01). Following bronchial provocation with allergen in nine subjects, plasma levels of adenosine increased from a mean base line of 5.4 +/- 0.9 to 9.6 +/- 2.0 ng/ml at 15 min (P less than 0.01) in parallel with a fall in forced expiratory volume in 1 s. With methacholine provocation bronchoconstriction reached maximum 2-5 min postchallenge being followed by, but not accompanied by, significant increases in plasma levels of adenosine. These data suggest that adenosine is a specific bronchoconstrictor that may contribute to airflow obstruction in asthma.     

The mechanism of the polynucleotide phosphorylase-catalyzed arsenolysis of ADP

Using ADP and arsenate (AsV), polynucleotide phosphorylase (PNPase) catalyzes the apparent arsenolysis of ADP to AMP-arsenate and inorganic phosphate, with the former hydrolyzing rapidly into AMP and AsV. However, in the presence of glutathione, AMP-arsenate may also undergo reductive decomposition, yielding AMP and arsenite (AsIII). In order to clarify the mechanism of ADP arsenolysis mediated by Escherichia coli PNPase, we analyzed the time course of the reaction in the presence of increasing concentrations of ADP, with or without polyadenylate (poly-A) supplementation. These studies revealed that increasing supply of ADP enhanced the consumption of ADP but inhibited the production of both AMP and AsIII. Formation of these products was amplified by adding trace amount of poly-A. Furthermore, AMP and AsIII production accelerated with time, whereas ADP consumption slowed down. These observations collectively suggest that PNPase does not catalyze the arsenolysis of ADP directly (in a single step), but in two separate consecutive steps: the enzyme first converts ADP into poly-A, then it cleaves the newly synthesized poly-A by arsenolysis. It is inferred that one active site of PNPase can catalyze only one of these reactions at a time and that high ADP concentrations favor poly-A synthesis, thereby inhibiting the arsenolysis.     

5'-Nucleotidase I from rabbit heart

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.     

The activation of non-phosphorylating electron transport by adenine nucleotides in Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

In isolated plant mitochondria the oxidation of both succinate and exogenous NADH responded in the expected manner to the addition of ADP or uncoupling agents, and the uncoupled rate of respiration was often in excess of the rate obtained in the presence of ADP. However, the oxidation of NAD+-linked substrates responded in a much more complex manner to the addition of ADP or uncoupling agents such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone to mitochondria oxidizing pyruvate plus malate failed to result in a reliable stimulation; this uncoupled rate could be stimulated by adding AMP or ADP in the presence of oligomycin or bongkrekic acid. Spectrophometric measurements showed that the addition of AMP or ADP resulted in the simultaneous oxidation of endogenous nicotinamide nucleotide and the reduction of cytochrome b. ADP was only effective in bringing about these changes in redox state in the presence of Mg2+ whereas AMP did not require Mg2+. It was concluded that AMP activated the flow of electrons from endogenous nicotinamide nucleotide to cytochrome b, possible at the level of the internal NADH dehydrogenase.