Metabolic fate of AMP, IMP, GMP and XMP in the cytosol of rat brain: an experimental and theoretical analysis

Abstract

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. Keywords: adenylic charge, differential equations, purine salvage, quantitative metabolism J. Neurochem. (2001) 76, 1291±1307.5 H -Nucleotidases (EC 3.1.3.5) catalyse the hydrolysis of purine or pyrimidine nucleoside 5 H -phosphates to the corresponding nucleosides and inorganic phosphate. An ecto 5 H -nucleotidase (e-N) (glycosyl-phosphatidylinositol (GPI)-anchored) and three forms appearing in the cytosolic subcellular fraction (e-Ns, c-N-I, c-N-II) have been described in vertebrates (for a review see Zimmermann 1992). Only two of these enzymes have been described in the cytosol of rat brain: e-Ns and c-N-II. e-Ns appears to be derived from the glycosyl phosphatidyl inositol (GP-I) anchored e-N (Orford and Saggerson 1996); it has a preference for AMP (low K m ) and is inhibited by ATP and ADP (K i , mm) and by EDTA (Ipata 1968;Mallol and Bozal 1983;Orford and Saggerson 1996). c-N-II has a preference for IMP and GMP over AMP. The enzyme puri®ed from rat brain (Marques et al. 1998) has molecular and kinetic characteristics similar to the enzyme from other sources: it is activated by dinucleoside polyphosphates (K a , mm, Pinto et al. 1986;Itoh and Yamada 1990), decavanadate (K a , nm, Le Hir 1991), ATP and ADP (K a , mM, Van den Berghe et al. 1977a; Itoh 1981). The enzyme from rat brain is also activated by polyphosphates (K a , mm, Marques et al. 1998). Depending on the extraction and assay procedures one of the two cytosolic 5 H -nucleotidases may pass unnoticed. In our experience, both enzymes can be detected in the cytosolic extracts of rat brain if the proper experimental conditions are set up.T...

H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae

“…The velocity equations considered for E 1 , E 3 , E 4 , E 5 , E 6 and E 7 were as in Torrecilla et al . [3]; those for E 2 and E 8 are indicated in the text and Table 3.…”

“…Aliquots were taken at the indicated times and analyzed by HPLC. In (B) application of the theoretical model was performed with the mathematica ‐3.0 program, as described in the text and in [3]. The V ‐values, from control cells, and the kinetic parameters described in Table 3 were used.…”

“…a behavior similar to that previously described for the AMP deaminase. Accordingly [3] the velocity equation used for 5′‐IMP nucleotidase was settled as: where n = 1.7–1.2[ATP]/( K a2ATP + [ATP]) and S 0.5 = K m2IMP – ( F2K [ATP]/( K a2ATP + [ATP])).…”

“…5) or 100 (Fig. 6) (see [3], and Table 3, for further explanations on the significance of these parameters).…”

Simultaneous Determination of Adenosine and Related Purines in Tissues and Hemolymph of Mussel by HPLC

“…In rat brain, it was observed that the formation of IMP in the intracellular space is predominant under normoxic or moderate hypoxic conditions, whereas the formation of adenosine prevails when ATP levels fall to anoxic values. [14,15] The accumulation of IMP is also predominant in skeletal muscle under hypoxia [16] and in flying muscle of locusts under anoxia. [17] Unlike the mammals, the animals adapted to hypoxia=anoxia transient are able to withstand without O 2 for long periods.…”