Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge

Barrette, William C.; Hannum, Diane M.; Wheeler, William D.; Hurst, James K.

doi:10.1128/jb.170.8.3655-3659.1988

Cited by 24 publications

(26 citation statements)

References 18 publications

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“…Elevated AMP and ADP levels lead to a significantly impaired energy charge of 0.68 in this cell line, whereas all other cell lines exhibited an energy charge between 0.7 and 0.8 which is within the physiological range obtained by in vivo experiments in different human and rat tissues and cell cultures [13,21,33]. In general, cells with energy charges greater than 0.7 to 0.8 divide and synthesize protein at near-maximal rates [34], suggesting that the lower energy charge in cd20 6 cells might lead to decreased growth rate. The greatly reduced growth rate of this cell line might also be a result of changes in AdoHcy and adenosine metabolism, because elevated AdoHcy levels decrease transmethylation activity [3][4][5] and high adenosine levels are known to induce cell death in several cell types [9][10][11].…”

Section: Discussionmentioning

confidence: 89%

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

Hermes¹,

Oßwald²,

Riehle³

et al. 2008

Cell Physiol Biochem

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Background/Aims: S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy), which is a potent product inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. While previous studies have shown that AdoHcyase inhibition or deficiency lead to a decreased AdoMet/AdoHcy ratio resulting in impaired transmethylation, the effect of enhanced AdoHcyase activity on AdoMet/AdoHcy metabolism and methylation reactions has not been studied in detail. Methods: To investigate the effect of enhanced AdoHcyase activity, we generated HEK-293 cell lines stably overexpressing AdoHcyase. Results: Initial studies revealed that 2-10-fold AdoHcyase overexpression resulted in decreased intracellular AdoHcy and elevated adenosine levels, whereas 16-fold AdoHcyase overexpression increased adenosine and AdoHcy levels, lowered energy charge, and altered cell morphology. Furthermore, we found a correlation between AdoHcyase activity and cell viability. Caspase-activity assays and DNA fragmentation analysis revealed that the cell death in AdoHcyase overexpressing cells was due to apoptosis. Global DNA methylation was not altered in the different AdoHcyase overexpressing cell lines. Conclusion: Taken together, these data show that 2-5-fold enhanced AdoHcyase activity is well tolerated by the cell, while greatly enhanced AdoHcyase activity results in adenosine-induced apoptosis. The fact that enhanced AdoHcyase activity does not increase transmethylation activity suggests that AdoHcyase activity under physiological conditions is not rate limiting for efficient transmethylation.

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Section: Discussionmentioning

confidence: 89%

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

Hermes¹,

Oßwald²,

Riehle³

et al. 2008

Cell Physiol Biochem

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“…Specifically, in R. rubrum under conditions identical to those used during collection of cells for determining the localization of P II to the membrane, the estimated energy charge is only about 0.66 and the ratio of ADP to ATP is approximately 1 within the limits of experimental error (38). Also, adenylate energy charge has been measured at significantly lower levels in both log and stationary-phase growth conditions in multiple bacterial species, where the ratio of ADP to ATP is as much as 3 (5,12,31,37). Given that nonlogarithmic growth conditions are more likely to be relevant in the wild, we predict that energy sensing by P II is an important function of this protein.…”

Section: Discussionmentioning

confidence: 93%

Specificity and Regulation of Interaction between the P _II and AmtB ₁ Proteins in Rhodospirillum rubrum

2007

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The nitrogen regulatory protein P II and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P II homolog, GlnJ, has higher affinity for an AmtB 1 -containing membrane than the other two P II homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB 1 complex in the presence of ATP and 2-KG, supporting a role for P II as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K d of 2-KG for GlnJ bound to AmtB 1 is 340 M, which is higher than that required for uridylylation of GlnJ in vitro, about 5 M. This supports a model where multiple 2-KG and ATP molecules must bind a P II trimer to stimulate release of P II from AmtB 1 , in contrast to the lower 2-KG requirement for productive uridylylation of P II by GlnD.The ammonium channel/rhesus (Amt/Rh) family of proteins is a widely distributed group of trimeric integral membrane proteins found in all domains of life that can function as gas channels of ammonia and perhaps carbon dioxide (13,20,29,39,43). A subset of this family, the AmtB proteins, is found in bacteria, archaea, some lower eukaryotes and plants. Homologs of amtB are often found in close proximity to genes encoding P II homologs (46). P II regulatory proteins are also found in most prokaryotes and some plant chloroplasts (3). P II is a small, soluble, trimeric protein that regulates the functions of several other proteins involved in nitrogen metabolism (3,4,35,36). AmtB appears to have two roles in the cell. The first function, to act as a channel for uncharged ammonia, has been explored physiologically, structurally, and computationally (28,33,44). The second function of AmtB is to interact with P II and has only recently been described (6,9,21,22,53).AmtB proteins have been shown to interact with homologs of P II in several bacteria and in the archaeon Methanococcus jannaschii (8,10,17,18,44,45,47,50,53). The association of P II with AmtB can physically block the ammonia gas channels of AmtB under conditions of nitrogen sufficiency in the cell. In addition, the AmtB-P II complex is able to recruit at least one other protein to the membrane, dinitrogenase reductase-activating glycohydrolase (DRAG), in organisms capable of nitrogen fixation (22,48). This membrane sequestration requires both an Amt protein and a P II protein and results in the inability of DRAG to activate dinitrogenase reductase in vivo. Finally, AmtB is able to remove equimolar amounts of P II from the cytoplasm, preventing P II from interacting with at least some other proteins. Although membrane sequestration of P II has b...

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“…In contrast, HEK-293 only exhibited an energy charge of 0.54. However, previous studies have shown, that the energy charge can be reduced to much lower values without apparent impairment of normal cellular function [47].…”

Section: Discussionmentioning

confidence: 99%

S-Adenosylhomocysteine Metabolism in Different Cell Lines: Effect of Hypoxia and Cell Density

Hermes

Hippel²,

Oßwald³

et al. 2005

Cell Physiol Biochem

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Background/Aims: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. Methods: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. Results: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 ± 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 ± 1.1 and 21.1 ± 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 ± 0.7 and 7.1 ± 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 ± 0.2 nmol min^-1 mg^-1 protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. Conclusions: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.

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Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge

Cited by 24 publications

References 18 publications

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

Specificity and Regulation of Interaction between the P _II and AmtB ₁ Proteins in Rhodospirillum rubrum

S-Adenosylhomocysteine Metabolism in Different Cell Lines: Effect of Hypoxia and Cell Density

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Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge

Cited by 24 publications

References 18 publications

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

S-Adenosylhomocysteine Hydrolase Overexpression in HEK-293 Cells: Effect on Intracellular Adenosine Levels, Cell Viability, and DNA Methylation

Specificity and Regulation of Interaction between the P II and AmtB 1 Proteins in Rhodospirillum rubrum

S-Adenosylhomocysteine Metabolism in Different Cell Lines: Effect of Hypoxia and Cell Density

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Specificity and Regulation of Interaction between the P _II and AmtB ₁ Proteins in Rhodospirillum rubrum