guanosine-triphosphate and adenylosuccinate

Since the standard Gibbs energies of formation are known for all the species in the purine nucleotide cycle at 298.15 K, the functions of pH and ionic strength that yield the standard transformed Gibbs energies of formation of the ten reactants can be calculated. This makes it possible to calculate the standard transformed Gibbs energies of reaction, apparent equilibrium constants, and changes in the binding of hydrogen ions for the three reactions at desired pHs and ionic strengths. These calculations are also made for the net reaction and a reaction that is related to it. The equilibrium concentrations for the cycle are calculated when all the reactants are initially present or only some are present initially. Since the concentrations of GTP, GDP, and P(i) may be in steady states, the equilibrium concentrations are also calculated for the system at specified steady-state concentrations.

Topics: Adenosine Monophosphate; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen-Ion Concentration; Osmolar Concentration; Phosphates; Purine Nucleotides; Thermodynamics

Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated.

Topics: Adenosine Monophosphate; Adenylosuccinate Synthase; Binding Sites; Cysteine; Escherichia coli; Fluorescent Dyes; Guanosine Triphosphate; Inosine Monophosphate; Kinetics; Ligands; Naphthalenesulfonates; Protein Conformation; Rhodamines; Spectrometry, Fluorescence

Nucleotide metabolism was studied in rats during and following the induction of 10 min of forebrain ischemia (four-vessel occlusion model). Purine and pyrimidine nucleotides, nucleotides, and bases in forebrain extracts were quantitated by HPLC with an ultraviolet detector. Ischemia resulted in a severe reduction in the concentration of nucleoside triphosphates (ATP, GTP, UTP, and CTP) and an increase in the concentration of AMP, IMP, adenosine, inosine, hypoxanthine, and guanosine. During the recovery period, both the phosphocreatine level and adenylate energy charge were rapidly and completely restored to the normal range. ATP was only 78% of the control value at 180 min after ischemic reperfusion. Levels of nucleosides and bases were elevated during ischemia but decreased to values close to those of control animals following recirculation. Both the decrease in the adenine nucleotide pool and the incomplete ATP recovery were caused by insufficient reutilization of hypoxanthine via the purine salvage system. The content of cyclic AMP, which transiently accumulated during the early recirculation period, returned to the control level, paralleling the decrease of adenosine concentration, which suggested that adenylate cyclase activity during reperfusion is modulated by adenosine A2 receptors. The recovery of CTP was slow but greater than that of ATP, GTP, and UTP. The GTP/GDP ratio was higher than that of the control animals following recirculation.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Brain; Chromatography, High Pressure Liquid; Cytidine Triphosphate; Guanine; Guanosine Diphosphate; Guanosine Triphosphate; Hypoxanthine; Hypoxanthines; Inosine; Inosine Monophosphate; Ischemic Attack, Transient; Male; Nucleotides; Phosphocreatine; Rats; Rats, Inbred Strains; Uridine Triphosphate

Topics: Adenosine Monophosphate; Amino Acids; Animals; Aspartic Acid; Cations, Divalent; Cations, Monovalent; Fatty Acids, Nonesterified; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inosine Nucleotides; Kinetics; Ligases; Male; Molecular Weight; Muscles; Nucleotides; Rats; Succinates