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

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.     

Diadenosine tetraphosphate activates cytosol 5'-nucleotidase

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver.     

Immunocytochemical localization of cytosol 5'-nucleotidase in chicken liver

We investigated the localization of cytosol 5'-nucleotidase in chicken liver by use of a pre-embedding immunoenzyme technique. Cytosol 5'-nucleotidase was purified from chicken liver and a monospecific antibody to this enzyme was raised in a rabbit. Fab fragments of the antibody were conjugated with horseradish peroxidase. Tissue sections of the fixed chicken liver were incubated with the peroxidase-Fab fragments, followed by DAB reaction for peroxidase. By light microscopy, dark-brown staining was present in the cytoplasm of parenchymal cells, Kupffer cells, and endothelial cells. The latter two types of cells were stained more strongly than the former. By electron microscopy, reaction deposits were present in the cytoplasmic matrix but not in cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes, or in nuclei. In control sections incubated with peroxidase-conjugated Fab fragments from non-immunized rabbit, no specific reaction was noted. The results indicate that cytosol 5'-nucleotidase is contained more in the sinus-lining cells and less in the parenchymal cells, and that the enzyme is present in the cytoplasmic matrix of these cells.     

Studies on some molecular properties of cytosol 5'-nucleotidase from rat liver

A cytosol 5'-nucleotidase was purified from rat liver. It appeared to be homogeneous on the criteria of polyacrylamide gel electrophoresis. We estimated the approximate molecular weight of the enzyme to be 200 000 and concluded that the enzyme most likely exists in the native state as a tetramer. Our results suggest that adenine nucleotides, which are activators of the enzyme, induce its conformational change.     

A reappraisal of the subunit molecular mass of chicken liver cytosol 5'-nucleotidase

The subunit molecular mass of chicken liver cytosol 5'-nucleotidase was earlier reported to be 51 kDa upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Naito, Y. and Tsushima, K. (1976) Biochim. Biophys. Acta 438, 159-168). By immunoblot analyses after SDS-gel electrophoresis, however, a fraction from the liver homogenized in the presence of leupeptin showed multiple bands with molecular masses around 57 kDa, and SDS-extracted proteins directly from the liver exhibited a single 70 kDa component. These results indicate that the 51 kDa peptide observed in the cytosol 5'-nucleotidase preparation might, in fact, be a proteolytic artifact.     

Purification and properties of AMP-deaminase from human uterine smooth muscle. Regulation by adenylate energy charge and activated fatty acids

At pH 7.0 and physiological concentrations of the main regulatory ligands (ATP, ADP, orthophosphate), human uterine muscle AMP-deaminase follows a hyperbolic type of saturation kinetics with S0.5 parameter value about 2 mM. The enzyme is regulated by adenylate energy charge (AEC) variations, being the most active at the AEC value 0.5-0.6 or 0.5-0.7, depending on the size of the total adenine nucleotide pool. Long-chain acyl-CoA strongly inhibit activity of the enzyme, influencing mainly the maximum velocity of the reaction.     

Hypoxia and the energy charge of the cerebral adenylate pool

A brief period of anoxia in vivo causes a transitory decrease in the size of the adenylate pool in the rat brain. This is probably caused by feedback inhibition by AMP of adenine nucleotide synthesis. Exposing rats to various degrees of hypoxia suggests that the sensitivity of the brain to lack of O(2) results from the brain's limited ability to maintain an adequate energy charge in unfavourable circumstances.     

Response of nucleoside diphosphate kinase to the adenylate energy charge

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.     

Stabilization of the adenylate energy charge by the depletion of adenylates without glycolytic stimulation

A relationship between the AMP deaminase reaction and the recovery of the energy charge was examined using permeabilized yeast cells. Citrate inhibited the glycolytic flux and the recovery of the energy charge. The addition of spermine enhanced the recovery of the energy charge without the reversal of the inhibition of glycolysis in the presence of excess citrate. The AMP deaminase reaction can participate in the stabilization of the energy charge only by the depletion of total adenylates, and not by the glycolytic stimulation under the conditions where citrate is highly increased during aerobic growth conditions.     

Corn phosphoglycolate phosphatase: Modulation of activity by pyridine nucleotides and adenylate energy charge

The activity of corn phosphoglycolate phosphatase (EC 3.1.3.18), a bundle sheath chloroplastic enzyme, is modulated, in vitro, both by NADP(H) and adenylate energy charge. The Vmax of the enzyme is increased by NADP (25%) and NADPH (16%) whatever the pH used, 7.0 or 7.9 respective pH of the stroma in the dark and in the light. At both pH, the adenylate energy charge alone has a positive effect with two peaks of activation, characteristics for this enzyme, at 0.2 and a maximum at 0.8 accentuated under nonsaturating concentration of phosphoglycolate. At low energy charge, NADP(H) increased the activation with an additive effect most particularly observed at pH 7.9 under saturating phosphoglycolate concentration; at high energy charge, NADP(H) had a positive or negative effect on the activation, depending on the pH value and the concentrations of substrate and NADP(H).The ferredoxin-thioredoxin system does not regulate the activity since i) DTT addition do not have any effect, ii) the light-reconstituted system containing ferredoxin, ferredoxin-thioredoxin reductase, thioredoxins and thylakoids is not effective either. However, light-dark experiments indicate that phosphophycolate phosphatase can be subjected to a fine tuning of its activity.All these data suggest that light cannot induce a modification of the protein but could exert a tight control of its activity by the intermediate of Mg²⁺ and substrate concentrations and the levels of metabolites such as NADP(H), ATP, ADP, AMP. So, the regulation of the activity shown, in vitro, by energy charge and NADP(H) might be of physiological significance.Abbreviations AEC adenylate energy charge - DTT dithiothreitol - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FTR ferredoxin-thioredoxin reductase - NADP-MDH NADP-malate dehydrogenase - P glycolate-phosphoglycolate - P glycolate phosphatase-phosphoglycolate phosphatase - PSII photosystem II - PPDK pyruvate, Pi dikinase - Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase