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.     

Human plasma gelsolin binds adenosine triphosphate

Binding studies of human plasma gelsolin with ATP were done by equilibrium dialysis. Analysis of the binding data showed that plasma gelsolin had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6. The bioluminescent assay for ATP with luciferin and firefly luciferase confirmed that the protein contained a nucleotide as ATP.     

Specific enzymatic determination of adenosine triphosphate

The well-known soluble kinases are not specific for ATP (1). All these enzymes convert ATP as well as GTP, ITP, CTP, and UTP, although at different rates. The only exception is adenylate kinase (1). However, with this enzyme, a direct determination of ATP in tissue extracts which contain both the di- and mononucleotides is not possible.Phosphoglycerate kinase from various sources is specific for ATP, GTP, and ITP and does not react with the pyrimidine nucleotides (2), Now, however, it was found that phosphoglycerate kinase from the blue alga Spirulina platensis does not convert GTP and ITP. With this enzyme, therefore, it is possible to specifically determine ATP in tissue extracts or in mixtures of nucleotides. In the same test, GTP and ITP can be determined by adding phosphoglycerate kinase from yeast or from other sources (2).     

Modification of sarcoplasmic reticulum adenosine triphosphatase by adenosine triphosphate magnesium

Lineweaver-Burk plots of Ca²⁺-activated adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum have been determined for a wide range of substrate concentrations. The plots measured at constant Mg²⁺ concentrations are normally nonlinear, but approach linearity either as the sarcoplasmic reticulum ages, or when small quantities of Triton-X100 are added. Titration with N-ethylmaleimide has the same effect on the activity of the ATPase measured either at high or low substrate concentrations. Lineweaver-Burk plots measured under conditions where the Mg²⁺ concentration is varied so as to be always equal to the ATP concentration are linear. These results have been interpreted as evidence that the adenosine triphosphatase has a single active site which uses MgATP as its substrate and which can be modified by free Mg²⁺.     

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.     

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.     

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.     

Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

The reaction between actomyosin and adenosine triphosphate

1. A study is made of the effect of adenosine triphosphate (ATP) upon the viscosity of solutions of actomyosin in 0.5 M KCl. 2. The observed effects are discussed in terms of an initial drop of the viscosity (viscosity response) and its subsequent slow reversal (recovery effect). The latter is ascribed to a decrease in the ATP concentration through enzymatic hydrolysis. 3. The recovery effect is inhibited by Mg, activated by Ca, in accordance with the effect of these ions on the activity of myosin-ATPase. 4. The viscosity response is not inhibited, probably promoted by Mg. It is not promoted, probably inhibited by Ca. 5. The viscosity response is induced not only by ATP, but to a certain extent also by inosinetriphosphate, inorganic triphosphate, and inorganic pyrophosphate, not by adenosine diphosphate or monophosphate. 6. The viscosity response could be obtained with enzymatically inactive myosin. 7. It is concluded that the effect of ATP upon myosin does not depend on its enzymatic hydrolysis.