Regulation of rat heart cytosol 5′-nucleotidase by adenylate energy charge

Abstract

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.

Purification of bovine liver cytosolic 5′‐nucleotidase

“…In our study we found that the sequential elution of glycoproteins from the concanavalin-A -Sepharose column by two competing sugars (methyl a-D-glucoside and methyl a-D-mannoside) permitted a considerable increase in the degree of purification of the 5'-nucleotidase. Hence, the enrichment factor of our preparation is higher than that obtained from rat liver [13], rat heart [20], chicken liver [50], bovine brain [15] or human placenta [48] enzymes. Our results suggest that the bovine cytosolic 5'-nucleotidase is a heterodimeric protein of about 130 kDa and differs in its structure from the membrane 5'-nucleotidase [30] which is a homodimeric protein (140 kDa).…”

“…The first one has the pyrimidine nucleotides as preferential substrates and the pH optimum of its activity occurs in a range between 7.5 and 9; the bovine liver and the human placental [48] enzymes belong to this group. The second one, including rat heart, and rat and chicken liver enzymes [13,14,20,511, is characterized by a maximal activity around pH 6.5 and a preferential affinity for the purine nucleotides and especially IMP.…”

“…Cation dependence of cytosolic 5'-nucleotidase 1 ml purified enzyme (total activity = 5 pmol/h) was applied at a flow rate of 30 ml/h for desalting on a Trisacryl GF05 column (1.14 x 3 cm; exclusion limit 3000) equilibrated with 40 mM Tris/HCl buffer pH 7.5. The enzyme activity was then measured in the absence or the presence of different cations (20 + strongly inhibit the purified cytosolic enzyme (Fig. 6), as they did for the plasma membrane enzyme [30].…”

Improvement by 5-Amino-4-Imidazole Carboxamide Riboside of the Contractile Dysfunction That Follows Brief Periods of Ischemia Through Increases in Ecto-5'-Nucleotidase Activity and Adenosine Release in Canine Hearts

“…25,34 Although ecto-5'-nucleotidase activity is affected by several neurohumoral and metabolic factors, 35 we measured 5'-nucleotidase in vitro, so it is likely that the active site of 5'-nucleotidase is directly affected by AICA riboside.…”

“…[7][8][9][10]35 Adenosine administered even after the onset of reperfusion attenuates the ischemia and reperfusion injury in the dogs, 9,10 although beneficial effects of adenosine administered during reperfusion may depend on the animal species and the duration of ischemia. 36,37 AICA riboside administered after the onset of myocardial ischemia did not improve the contractile dysfunction in the dogs.…”