Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.     

Effect of dipyridamole on inosine triphosphate pyrophosphohydrolase activity and inosine triphosphate content in fresh human erythrocytes incubated with adenosine

The activity of inosine triphosphate pyrophosphohydrolase (ITPH) in human erythrocytes was found to be 1.50 +/- 0.39 mumol of inosine triphosphate (ITP) hydrolysed x min-1 per g Hb, and no measurable amount of ITP was detected. When dipyridamole was added to the medium composed of adenosine, pyruvate and inorganic phosphate, ITPH activity was 1.18 +/- 0.41, and at the same time ITP accumulation was 0.61 +/- 0.31 mumol/g Hb. The negative correlation between ITPH activity and accumulation of ITP was r = -0.87 at P less than 0.001.     

Spontaneous reactivation of covalently labeled proton adenosinetriphosphatase

Bovine heart mitochondrial adenosinetriphosphatase selectively labeled by [14C]-N,N'-dicyclohexylcarbodiimide or [14C]-7-chloro-4-nitro-2,1,3-benzoxadiazole was used together with other components to form reconstituted submitochondrial particles. When assayed for ATP hydrolysis under normal hydrolysis condition, these labeled submitochondrial particles were found to increase slowly in specific activity with preincubation time, without losing the covalent label. But when assayed for oxidative phosphorylation, the ratio of the specific activity of the same labeled particles to that of the control particles was higher and was unaffected by preincubation. If the labeled particles had been treated by a simulated procedure for oxidative phosphorylation measurement before the ATPase assay, their specific activities for ATP hydrolysis were also found to be higher and unaffected by preincubation. These observations are difficult to reconcile with the alternating three-site model for proton adenosinetriphosphatase or any model which requires the sequential action of three identical sites for ATP hydrolysis and synthesis. A new model with one active and two latent interacting sites is proposed for interpreting the present data.     

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.     

Localization of pyridoxal-5-phosphate, covalently bound with human hemoglobin. Spectrofluorimetric studies]

Human apohemoglobin tryptophan residues were localized in the regions of the protein globule with restricted mobility. By the method of dynamic quenching of phosphopyridoxyl chromophore fluorescence, the heterogeneity of pyridoxal-5-phosphate molecules covalently bound to the human hemoglobin molecules was determined from the accessibility to solvent. The first four pyridoxal-5-phosphate molecules are localized in the hydrophobic regions of the hemoglobin molecule; at the same time, they have a high mobility. One of these molecules is situated at the site inaccessible to the solvent, which coincides with the anion-binding center of the oxyhemoglobin molecule. The next pyridoxal-5-phosphate molecules modify the surface amino groups of the protein. In the apohemoglobin molecule, the pyridoxal-5-phosphate binding sites are more exposed to the solvent, as compared to hemoglobin. In the hemoglobin molecule modified by pyridoxal-5-phosphate, an effective electron excitation energy transfer from tryptophan residues to phosphopyridoxyl chromophores occurs. The effective distances between tryptophanyls of single subunits of hemoglobin and the covalently bound pyridoxal-5-phosphate molecule were estimated to be 19 A for the alpha-subunit and 17 A for the beta-subunit.     

Ticagrelor induces adenosine triphosphate release from human red blood cells

RationaleThe novel P2Y₁₂ antagonist ticagrelor inhibits ADP-induced platelet aggregation more rapidly and more potently than clopidogrel. Clinical trials have revealed dyspnea and asymptomatic ventricular pauses as side effects of ticagrelor. The mechanism behind these side effects is not known, but it is plausible that they are mediated by adenosine.ObjectiveTicagrelor is known to increase adenosine concentrations by inhibiting red blood cell reuptake, but the potency of this effect may be too low to fully explain the adenosine related effects. The purpose of the present study was to determine whether ticagrelor has other effects on red blood cells (RBCs) that could contribute to explain the pleiotropic effects seen with ticagrelor treatment.Methods and resultsUsing a luciferase-based bioluminescence assay, we studied ATP release in human blood. Human RBCs responded to ticagrelor in vitro by releasing substantial amounts of ATP in a dose-dependent manner (IC₅₀ 14 μM). The rapid effect indicates release through membrane channels, which was supported by a depolarizing effect of ticagrelor and inhibition of ATP release by anion channel blockers.ConclusionIn conclusion, our data show that, in vitro, ticagrelor can induce ATP release from human RBCs, which is subsequently degraded to adenosine. Further studies are warranted to determine what role this mechanism may play in the clinical effects of ticagrelor.