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Research paper thumbnail of Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis: Nucleotide metabolism in rat brain cytosol

Journal of Neurochemistry, Mar 1, 2001

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytoso... more A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P i generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V max and K m values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of MATHEMATICA 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the in¯uence of nM±mM concentrations of ATP on the metabolism of AMP.

Research paper thumbnail of Labeled adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N). Synthesis with firefly luciferase

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important m... more Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(S)tetraphosphdS')adenosine (Ap,A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap,A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [j3,r-32PjATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap,A is obtained. Radioactive adenosine(S)tetraphospho(S)nucleoside (Ap,N) can also be synthesized from ATPyS and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive Ap,A and pnA (n > 4) can also be approached with luciferase.

Research paper thumbnail of Uric acid synthesis by rat liver supernatants from purine bases, nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in re... more The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1mM concentration (except xanthine, 0.5 mM and guanosine and guanine, 0.1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g-' of wet liver min-' were:

Research paper thumbnail of Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: An experimental and theoretical analysis

Journal of …, 2001

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytoso... more A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was ...

Research paper thumbnail of Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis: Nucleotide metabolism in rat brain cytosol

Journal of Neurochemistry, Mar 1, 2001

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytoso... more A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was preferentially degraded to adenosine with concomitant appearance of inosine and hypoxanthine. In the presence of ATP, AMP was preferentially degraded via IMP. The nucleosides generated in the course of the reactions are further degraded, almost exclusively, via nucleoside phosphorylase using as cofactor the P i generated in the reaction mixture. In order to quantify the effect of each one of the enzymes involved in the degradation of NMP, two complementary approaches were followed: (i) the V max and K m values of the enzymes acting in the intermediate steps of the reactions were determined; (ii) these data were introduced into differential equations describing the concentration of the nucleotides and their degradation products as a function of the time of incubation. Factors affecting kinetic parameters of the equation velocity as a function of ATP concentration were introduced when required. The differential equations were solved with the help of MATHEMATICA 3.0. The theoretical method can be used to simulate situations not feasible to be carried out, such as to measure the in¯uence of nM±mM concentrations of ATP on the metabolism of AMP.

Research paper thumbnail of Labeled adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N). Synthesis with firefly luciferase

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important m... more Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(S)tetraphosphdS')adenosine (Ap,A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap,A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [j3,r-32PjATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap,A is obtained. Radioactive adenosine(S)tetraphospho(S)nucleoside (Ap,N) can also be synthesized from ATPyS and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive Ap,A and pnA (n > 4) can also be approached with luciferase.

Research paper thumbnail of Uric acid synthesis by rat liver supernatants from purine bases, nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in re... more The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1mM concentration (except xanthine, 0.5 mM and guanosine and guanine, 0.1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g-' of wet liver min-' were:

Research paper thumbnail of Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: An experimental and theoretical analysis

Journal of …, 2001

A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytoso... more A systematic study of the metabolic fate of AMP, IMP, GMP and XMP (NMP) in the presence of cytosol from rat brain is here presented; the kinetics of both disappearance of NMP, and appearance of their degradation products was followed by HPLC. In the absence of ATP, AMP was ...