Response of the intracellular adenosine triphosphate pool of Saccharomyces cerevisiae to growth inhibition induced by excess L-methionine

Yeast cells accumulate S-adenosyl-l-methionine (S-AM) when cultivated in the presence of l-methionine. Cell growth is inhibited by the addition of high concentrations of l-methionine. A number of investigators have attributed this to the depletion of adenosine triphosphate (ATP) as a consequence of the utilization of that mucleotide for S-AM formation. The cellular ATP pool of Saccharomyces cerevisiae was measured during growth inhibition caused by addition of excess l-methionine. Polyethylenimine thin-layer chromatography and subsequent autoradiography were used to quantitate the extracted ATP. Addition of l-methionine to a level of 5 mg/ml in a culture during exponential growth caused an increase in the doubling time of 40 to 50%. During this period, the cellular ATP level continued increasing normally and, as the cells entered stationary growth, receded to a level characteristic of an uninhibited stationary culture growth. After the addition of methionine, there was never an observed depletion of the ATP pool other than the normal fluctuation which occurs in an uninhibited culture. We have concluded that growth inhibition by excessive methionine does not result from limiting availability of ATP.     

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.     

Probes of catalytic site cooperativity during catalysis by the chloroplast adenosine triphosphate and the adenosine triphosphate synthase

During net nucleoside triphosphate synthesis by chloroplast ATP synthase the extent of water oxygen incorporation into each nucleoside triphosphate released increases with decrease in ADP, GDP or IDP concentration. Likewise, during net ATP hydrolysis by the Mg2+-activated chloroplast ATPase, the extent of water oxygen incorporation into each Pi released increases as the ATP, GTP, or ITP concentration is decreased. However, the concentration ranges in which substrate modulation occurs differs with each nucleotide. Modulation of oxygen exchange during synthesis and hydrolysis of adenine nucleotides, as measured by variation in the extent of water oxygen incorporation into products, occurs below 250 microM. In contrast, guanosine and inosine nucleotides alter the extent of exchange at higher and much wider concentration ranges. Activation of the chloroplast ATPase by either heat or trypsin results in similar catalytic behavior as monitored by ATP modulation of oxygen exchanges during hydrolysis in the presence of Mg2+. More exchange capacity is evident with octylglucoside-activated enzyme at all ATP concentrations. High levels of tentoxin were also found to alter the catalytic exchange parameters resulting in continued water oxygen exchange into Pi released during hydrolysis at high ATP concentrations. Little or no oxygen exchange accompanies ATP hydrolysis in the presence of Ca2+. The [18O]Pi species formed from highly gamma-18O-labeled ATP at lower ATP concentrations gives a distribution as expected if only one catalytic pathway is operative at a given ATP concentration. This and other results support the concept of catalytic cooperativity between alternating sites as explanation for the modulation of oxygen exchange by nucleotide concentration.     

The polymerization of proteins; adenosine triphosphate and the polymerization of actin

It was found that actin solutions contain ATP. Experiments in which hexokinase and creatine were added to actin show that the ensuing dephosphorylation of ATP affects actin so that it will not polymerize.     

Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin–Al3+ complex and its analytical application

A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)–aluminium (III) (Al³⁺) complex and ATP and studied using UV‐visible and fluorescence spectroscopy. Weak luminescence spectra of Al³⁺ were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al³⁺ ion. It was observed that the FL emission spectrum of GFLX–Al³⁺ was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10^–10 – 1.0 × 10^–8 mol L^–1 with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10^–11 mol L^–1 for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10^–8 mol L^–1 ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Intracellular compartmentalization of adenosine triphosphate

The intracellular distribution and diffusivity of adenosine triphosphate (ATP) was studied by cryomicrodissection of individual Rana pipiens oocytes. We measured ATP concentrations in the nucleus, in animal and vegetal hemisphere cytoplasm, and in an intracellular reference phase (iRP, a microinjected gelatin "organelle") which samples diffusive ATP. Regional concentrations were not equal: nucleus much greater than animal ooplasm greater than vegetal ooplasm. ATP binding and water availability (as solvent) were determined by plotting nuclear and cytoplasmic ATP concentrations as a function of reference phase ATP concentrations (isothermal analysis). The nucleus/iRP isotherm for ATP was an equimolar line, showing that nucleoplasm resembles iRP gelatin (and consequently a simple aqueous solution) in its solvent properties. Cytoplasm/iRP isotherms were more complex, having slopes much less than unity and ordinal intercepts above the graph's origin. They demonstrate the presence in cytoplasm of mechanisms that are capable of excluding and binding ATP. These mechanisms are responsible for the inhomogeneity in ATPs intracellular distribution. In addition, exclusion and binding have different and opposing effects on ATP concentrations in the cell's "soluble space," and hence on ATP availability to enter into cellular reactions. It follows that these phenomena must be considered in attempts to model ATPs role in metabolism.     

Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase.

Radioactive adenosine triphosphate was synthesized transiently from adenosine diphosphate and radioactive inorganic phosphate by sodium and potassium adenosine triphosphatase from guinea pig kidney. In a first step, K+-sensitive phosphoenzyme was formed from radioactive inorganic phosphate in the presence of magnesium ion and 16 mM sodium ion. In a second step the addition to the phosphoenzyme of adenosine diphosphate with a higher concentration of sodium ion produced adenosine triphosphate. Recovery of adenosine triphosphate from the phosphoenzyme was 10 to 100% in the presence of 96 to 1200 mM sodium ion, respectively. Potassium ion (16mM) inhibited synthesis if added before or simultaneously with the high concentration of sodium ion but had no effect afterward. The half-maximal concentration for adenosine diphosphate was about 12 muM. Ouabain inhibited synthesis. The ionophore gramicidin had no significant effect on the level of phosphoenzyme nor on the rate nor on the extent of synthesis of adenosine triphosphate. The detergent Lubrol WX reduced the rate of phosphoenzyme break-down and the rate of synthesis but did not affect the final recovery. Phospholipase A treatment inhibited synthesis. In a steady state, the enzyme catalzyed a slow ouabain-sensitive incorporation or inorganic phosphate into adenosine triphosphate. These results and other suggest that binding of sodium ion to a low affinity site on phosphoenzyme formed from inorganic phosphate is sufficient to induce a conformational change in the active center which permits transfer of the phosphate group to adenosine diphosphate.     

A portrait of the adenosine triphosphate synthetase-hydrolase

It is proposed that the ATP-synthetic and ATP-hydrolytic activities of energy-transducing mitochondria, chloroplasts and bacterial membranes are due to different enzyme systems. It is suggested that the alpha-subunits of the oligomycin-sensitive coupling factor catalyze synthesis and the beta-subunits catalyze hydrolysis. Evidence is assembled from the literature in support of this concept.     

Cosignaling of adenosine and adenosine triphosphate in hypobaric hypoxia-induced hypothermia

Purines, that is, adenosine and ATP, are not only products of metabolism but are also neurotransmitters. Indeed, purinergic neurotransmission is involved in thermoregulatory processes that occur during normoxia. Exposure to severe hypoxia elicits a sharp decrease in body core temperature (T(CO)), and adenosinergic mechanisms have been suspected to be responsible for this hypothermia. Because ATP per se and its metabolite adenosine could have complex interactions in some neural networks, we hypothesize that both adenosine and ATP are involved in the central mechanism of hypoxia-induced hypothermia. Their role in the thermoregulatory process was therefore investigated in a 24-h hypobaric hypoxia (Fi(O2) = 10%), using CGS-15943, a nonselective antagonist of adenosine receptors, and suramin, an ATP receptor antagonist. T(CO) and spontaneous activity (A(S)) were monitored by telemetry in conscious rats, receiving CGS-15943 (10 mg/kg ip), suramin (7 nmol icv), or both. The same treatments were done in normoxia to evaluate the specificity of their thermoregulatory action observed in hypoxia. Suramin/CGS-15943 treatment blunted the profound hypothermia observed in control rats throughout the hypoxia exposure, whereas CGS-15943 treatment blunted hypothermia during only 3 h, and suramin treatment had no effect. These results suggest that suramin potentiates the CGS-15943 effects and consequently that adenosine and ATP signaling act in synergy. In normoxia, suramin/CGS-15943 induced an increase in T(CO) but to a far lesser extent than observed in hypoxia. Thus it might be suggested that the suramin/CGS-15943 blunting of hypoxia-induced hypothermia would be specific to hypoxia-induced mechanisms.