Ion Levels and Membrane Potential in Chick Heart Tissue and Cultured Cells

Intracellular concentrations of sodium and potassium as well as resting potentials and overshoots have been determined in heart tissue from chick embryos aged 2–18 days. Intracellular potassium declined from 167 mM at day 2 to 117–119 mM at days 14–18. Intracellular sodium remained nearly constant at 30–35 mM during the same period. The mean resting potential increased from -61.8 mV at day 3 to about -80 mV at days 14–18. The mean overshoot during the same period increased from 12 to 30 mV. P_Na/P_K calculated from the ion data and resting potentials declined from 0.08 at day 3 to 0.01 at days 14–18. Thus, the development of embryonic chick heart during days 2–14 is characterized by a declining intracellular potassium concentration and an increasing resting potential and overshoot. Heart cells from 7- to 8-day embryos, cultured either in monolayer or reassociated into aggregates, were compared with intact tissue of the same age. The intracellular concentrations of sodium and potassium were similar in the three preparations and cultured cells responded to incubation in low potassium medium or treatment with ouabain in a manner similar to that of intact tissue. Resting potentials and overshoots were also similar in the three preparations.     

The effect of temperature on veratridine action in squid giant axons

Resting membrane potential and intracellular sodium and potassium concentrations were determined at 5 and 21°C in normal and veratridine-treated axons of the squid Doryteuthis plei. 300 μM veratridine produced an increase in the intracellular sodium concentration, which changed from 52 to 284 mM in 10 min of exposure at 21°C, and from 76 to 260 mM at 5°C. Under the same treatment the intracellular potassium concentration changed from 357 to 221 mM (21°C) and from 334 to 194 mM (5°C). All the changes could be prevented by adding 1 μM tetrodotoxin. Veratridine (30, 100 and 300 μM) increased the resting sodium permeability of the giant axon, and the effect was greater at 21°C. The affinity of the membrane for veratridine increases when the nerves are cooled, the three concentrations tested produce maximum activation of the sodium channels at 5°C. But only the higher two concentrations are saturating at 21°C.     

Relative Ion Permeabilities in the Crayfish Giant Axon Determined from Rapid External Ion Changes

The changes in membrane potential of isolated, single crayfish giant axons following rapid shifts in external ion concentrations have been studied. At normal resting potential the immediate change in membrane potential after a variation in external potassium concentration is quite marked compared to the effect of an equivalent chloride change. If the membrane is depolarized by a maintained potassium elevation, the immediate potential change due to a chloride variation becomes comparable to that of an equivalent potassium change. There is no appreciable effect on membrane potential when external sodium is varied, at normal or at a depolarized membrane potential. Starting from the constant field equation, expressions for the permeability ratios P _Cl/P _K, P _Na/P _K, and for intracellular potassium and chloride concentrations are derived. At normal resting membrane potential, P _Cl/P _K is 0.13 but at a membrane potential of -53 mv (external potassium level increased about five times) it is 0.85. The intracellular concentrations of potassium and chloride are estimated to be 233 and 34 mM, respectively, and it is pointed out that this is not compatible with ions distributed in a Nernst equilibrium across the membrane. It is also stressed that the information given by a plot of membrane potential vs. the logarithm of external potassium concentrations is very limited and rests upon several important assumptions.     

Use of low temperature and high K+ incubation media for in vitro tissue preparation for X-ray microanalysis

Incubation of tissue slices in physiological buffers gives rise to significant changes in the intracellular ion concentrations, which may disturb subsequent X-ray microanalysis. In the present study it was attempted to design incubation conditions that retain the in vivo conditions better. The following variables were investigated: (1) exchange of Na⁺ in the incubation medium for K⁺, and exchange of Cl^–for the less permeable gluconate anion; (2) incubation at 4°C rather than at 37°C; and (3) addition of dextran to the incubation medium. Brief exposure (a few seconds) of liver slices to a buffer causes changes in the intracellular Na, Cl and K concentrations, depending on the ionic composition of the buffer. Incubation in a normal physiological (high NaCl) buffer at 37°C results in a further increase of Na and Cl and a further decrease in K in liver cells. The changes reach a maximum at 30 min and the concentrations then remain stable throughout a 2-h incubation. Incubation in sodium gluconate medium or addition of dextran to the physiological buffer somewhat reduces the changes in the intracellular ion composition (compared to the standard physiological incubation medium). Incubation in potassium gluconate medium results in a decrease in cellular Na and an increase in K. Quantitative morphological studies show that tissue oedema is observed to the same extent in hepatocytes incubated in sodium gluconate, potassium gluconate and physiological buffer containing 10% dextran. However, these buffers cause significantly less cell oedema than the physiological (high NaCl) buffer. Incubation of liver, cerebral cortex or submandibular gland slices in physiological (high NaCl) solutions at 4°C for 4 h caused a more extensive increase in Na⁺ and decrease in K⁺ than incubation at 37°C for 2 h. This suggests inhibition of the Na⁺, K⁺-ATPase under these conditions. As compared to incubation at 37°C for 2 h, tissues incubated in potassium gluconate buffer at 4°C for 4 h have a cellular K concentration closer to the in situ value. Cholinergic stimulation of tissue slices from cerebral cortex and submandibular gland at room temperature for 1 min shows the best physiological response in tissue slices preincubated at 4°C for 4 h in high KCl, potassium gluconate and high NaCl, in this order. The response can, however, only be seen, when cholinergic stimulation is carried out in a standard physiological buffer with a high NaCl concentration. It is concluded that in vitro storage of tissue for X-ray microanalysis is best carried out at 4°C in a solution with a high K⁺ concentration.     

Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

The use of hyperosmolar,intracellular-like solutions for the isolation of epithelial cells from guinea-pig small intestine

Isolated small intestinal epithelial cells were prepared by using either (a) hyperosmolar, low sodium, high potassium containing (intracellular-like) solutions, or (b) isoosmolar, high sodium, low potassium containing (extracellular-like) solutions. Both (a) and (b) cells show high viability as estimated by Trypan blue exclusion, oxygen consumption, cellular ATP content, lactate-dehydrogenase liberation, intracellular ion concentrations and significant Na⁺-dependent alanine and uridine uptakes. Although (a) and (b) cells show in the cold similar ion concentration, after reincubation at 37° C for 30 min (a) cells show intracellular ion concentrations of 31 mM Na, 129 mM K and 88 mM Cl, whilst (b) cells have 71 mM Na, 93 mM K and 102 mM Cl. Cells prepared with (a) concentrate much more alanine and uridine than cells prepared with (b), probably because the latter have a lower Na⁺ gradient across the plasma membrane. Cells prepared with intracellular-like solutions would be an ideal system to study Na⁺-dependent transport mechanisms and the regulatory systems of intracellular ion concentrations.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Properties of single potassium channels in guinea pig hepatocytes

The patch-clamp technique of cell-attached and inside-out configurations was used to study the single potassium channels in isolated guinea pig hepatocytes. The single potassium channels in isolated guinea pig hepatocytes were recorded at different K⁺ concentrations. A linear single-channel current-voltage relationship was obtained at the voltage range of -80 to -20 mV with slope conductance of 70 ± 6 pS (n = 10). Under symmetrical high K⁺ concentration of 148 mM in the cell-attached patch membrane, the I-V curve exhibited a mild inward rectification at potentials positive to +20 mV. The values of reversal potential was +5 ± 2 mV (n = 10). When the external potassium concentration ([K⁺]₀) was decreased to 74 mM and 20 mM, the slope conductance was decreased to 48 ± 2 pS (n = 4) and 24 ± 3 pS (n = 3), respectively. The reversal potential was changed by 58 mV for a tenfold change in [K⁺]₀, indicating that this channel was highly selective for K⁺. Open probabilities (P₀) of the channel were 73-93% without apparent voltage dependence. The distributions of open time of the channels were fitted to two exponentials, while those of closed time were fitted to three exponentials, exhibiting no voltage dependence. The success rate of K⁺ channel activity to be recorded was 28% at room temperature, and there were no increases in the success rate nor in the channel opening probabilities at a temperature of 34-36°C. P₀ in inside-out patches was not changed by application of 1 μM Ca²⁺ nor 1 mM Mg²⁺ to the internal side of patch membranes. It is concluded that a novel type of the K⁺ channels in guinea pig hepatocytes had different properties of slope conductance, channel kinetics, and sensitivity to [Ca²⁺]_i, from those in other species. © 1994 Wiley-Liss, Inc.     

Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Summary To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands ofSqualus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na₂SO₄, 25 mM K₂SO₄ gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide. 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na₂SO₄, 50 mM K₂SO₄ gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.Abbreviations SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TALH thick ascending limb of Henle's loop     

Potassium activation of Na+-dependent leucine transport in brush-border membrane vesicles from rat jejunum

Na⁺-dependent leucine uptake was greater in potassium loaded brush-border membrane vesicles compared with controls. This effect was not mediated by an electrical potential difference, since it was still present in voltage-clamped conditions. Inhibition experiments indicate the same Na⁺-dependent leucine transport activity in the presence or in the absence of potassium. The affinity of sodium for the cotransporter was identical at 10 or 100 mM potassium. Leucine kinetics at different potassium concentrations showed a maximum 2.4-fold increase in V_max, while K_m was unaffected. The secondary plots of the kinetic results were not linear. This kinetic behaviour suggests that K⁺ acts as a non-essential activator of Na⁺-dependent leucine cotransport. A charge compensation of sodium-leucine influx is most probably a component of the potassium effect in the presence of valinomycin.     

Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.     

Changes in the Membrane Permeability of Frog's Sartorius Muscle Fibers in Ca-Free EDTA Solution

The changes in the membrane permeability to sodium, potassium, and chloride ions as well as the changes in the intracellular concentration of these ions were studied on frog sartorius muscles in Ca-free EDTA solution. It was found that the rate constants for potassium and chloride efflux became almost constant within 10 minutes in the absence of external calcium ions, that for potassium increasing to 1.5 to 2 times normal and that for chloride decreasing about one-half. The sodium influx in Ca-free EDTA solution, between 30 and 40 minutes, was about 4 times that in Ringer's solution. The intracellular sodium and potassium contents did not change appreciably but the intracellular chloride content had increased to about 4 times normal after 40 minutes. By applying the constant field theory to these results, it was concluded that (a) P_Cl did not change appreciably whereas P_K decreased to a level that, in the interval between 10 and 40 minutes, was about one-half normal, (b) P_Na increased until between 30 and 40 minutes it was about 8 times normal. The low value of the membrane potential between 30 and 40 minutes was explained in terms of the changes in the membrane permeability and the intracellular ion concentrations. The mechanism for membrane depolarization in this solution was briefly discussed.     

Electrophysiology of phagocytic membranes: intracellular K+ activity and K+ equilibrium potential in macrophage polykaryons

The role of K⁺ as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca²⁺ injections into macrophage polykaryons is studied. The intracellular K⁺ activity (a_K) and the K⁺ equilibrium potential (E_K) are measured using ion-sensitive microelectrodes. The mean value of a_K is 84 ± 5 mM in a culture medium containing 5.3 mM K⁺, but increases to 100 ± 8 mM when the extracellular K⁺ concentration is raised to 30.3 mM. Under the same conditions the values of E_K obtained from the Nernst equation are −81 ± 2 mV and −40 ± 2 mV, respectively. The reversal potentials (E_R) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean E_R values obtained are −74 ± 8 mV in the presence of low K⁺ concentration and −37 ± 3 mV for the high K⁺ medium. These values are significantly smaller than the estimated E_K for the corresponding situations. Evidence for the existence of an electrogenic (Na⁺ + K⁺)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.     

On the Concept of Resting Potential—Pumping Ratio of the Na+/K+ Pump and Concentration Ratios of Potassium Ions Outside and Inside the Cell to Sodium Ions Inside and Outside the Cell

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na⁺/K⁺ pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na⁺/K⁺ pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na⁺/K⁺ pump to prove that the three Na⁺ to two K⁺ pumping ratio of the Na⁺/K⁺ pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na⁺/K⁺ pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is ?30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na⁺/K⁺ pump and the ion concentration ratios play a role in the evolution of animal cells.     

Current Separations in Myxicola Giant Axons

The effect of reducing the external sodium concentration, [Na]_o, on resting potential, action potential, membrane current, and transient current reversal potential in Myxicola giant axons was studied. Tris chloride was used as a substitute for NaCl. Preliminary experiments were carried out to insure that the effect of Tris substitution could be attributed entirely to the reduction in [Na]_o. Both choline and tetramethylammonium chloride were found to have additional effects on the membrane. The transient current is carried largely by Na, while the delayed current seems to be independent of [Na]_o. Transient current reversal potential behaves much like a pure Nernst equilibrium potential for sodium. Small deviations from this behavior are consistent with the possibility of some small nonsodium component in the transient current. An exact P_Na/P_K for the transient current channels could not be computed from these data, but is certainly well greater than unity and possibly quite large. The peak of the action potential varied with [Na]_o as expected for a sodium action potential with some substantial potassium permeability at the time of peak. Resting membrane potential is independent of [Na]_o. This finding is inconsistent with the view that the resting membrane potential is determined only by the distribution of K and Na, and P_Na/P_K. It is suggested that P_Na/P_K's obtained from resting membrane potential-potassium concentration data do not always have the physical meaning generally attributed to them.     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.