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Cheryl A Hillery - One of the best experts on this subject based on the ideXlab platform.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Ca2+-calmodulin (Ca2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate Adenine Nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total Adenine Nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0.05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca2+-CaM activates AMPD in SCD. Finally, Adenine treatment (100 micromol/l) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)-exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0.05 compared to untreated samples). We conclude that Ca2+-CaM activation of AMPD contributes to increased turnover of the Adenine Nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Erythrocytes are unique among human tissues and cells because they do not contain enough adenylosuccinate synthetase activity to sustain measurable anabolic flow through the inosine monophosphate (IMP) to adenosine monophosphate (AMP) branch point of purine Nucleotide biosynthesis (Bishop, 1960; Lowy & Dorfman, 1970). The immediate metabolic consequence of this non-functional IMP to AMP branch point pathway is an inability to synthesize Adenine Nucleotides from either the de novo pathway or from the salvage pathway for hypoxanthine, the most abundant purine compound in the circulation (see Fig S1). Although enzymes are present for the salvage synthesis of AMP directly from adenosine and Adenine, the circulating levels of both compounds are normally quite low, i.e., <1 µmol/1 (Ericson et al, 1980; Moser et al, 1989). Consequently, erythrocytes have a severely limited capacity for maintaining their Adenine Nucleotide pool and the associated cellular energy reserves. This is particularly evident under conditions of energy imbalance that result in a net turnover of ATP, which can lead to activation of catabolic enzymes and an accelerated loss of Adenine Nucleotides. AMP deaminase (AMPD) converts AMP to IMP, a catabolite that has no anabolic route back into the erythrocyte Adenine Nucleotide pool (Fig S1). Thus, it has long been recognized that erythrocytes attempt to preserve their Adenine Nucleotide pool by maintaining an intracellular environment that minimizes AMPD catalytic activity (Askari, 1966). Several small molecule inhibitors of erythrocyte AMPD have been identified, including inorganic phosphate (Askari, 1966, Lian & Harkness, 1974; Yun & Suelter, 1978), 2,3-diphosphoglycerate (Askari & Rao, 1968; Lian & Harkness, 1974; Yun & Suelter, 1978), and phosphatidylinositol 4,5-bisphosphate (Sims et al, 1999), with measured Ki values that are similar to estimated intracellular concentrations in erythrocytes (Bontemps et al, 1986). The inherent capacity of normal erythrocytes to maintain Adenine Nucleotides during an energy crisis is reflected by the accumulation of AMP, rather than IMP, during short periods of accelerated ATP turnover in response to glucose deprivation (Bontemps et al, 1986; Mahnke & Sabina, 2005). Conversely, IMP, rather than AMP, accumulates in calcium-permeabilized erythrocytes (Almarez et al, 1988; Engstrom et al, 1996). The underlying mechanism responsible for rapid IMP accumulation under these conditions was not immediately evident because calcium activation of AMPD was lost upon dilution of the haemolysate or after the enzyme was partially purified (Almarez & Garcia-Sancho, 1989). This confounding issue was subsequently clarified by the discovery that calcium-calmodulin (Ca2+-CaM) activates erythrocyte AMPD and overcomes the inhibitory mechanisms inherent to these cells (Mahnke & Sabina, 2005). Similar Adenine Nucleotide metabolic dysregulation exists in clinical disorders of disturbed erythrocyte calcium homeostasis. For example, familial phosphofructokinase deficiency (FPD) is accompanied by a mild compensated anaemia and erythrocytes isolated from these individuals contain increased intracellular calcium (Waldenstrom et al, 2001) and a smaller Adenine Nucleotide pool concomitant with elevated levels of IMP (Ronquist et al, 2001; Sabina et al, 2006). The slowing of additional IMP accumulation during an experimental period of energy imbalance when FPD erythrocytes were pre-incubated with a diffusible CaM antagonist (Sabina et al, 2006) provided evidence that the protein–protein interaction between erythrocyte AMPD and Ca2+-CaM contributes to this metabolic dysregulation. Intracellular calcium is also elevated in sickle erythrocytes (Eaton et al, 1973; Palek, 1977; Bookchin & Lew, 1980), where it is primarily sequestered into intracellular vesicles by an inside-out ATPase pump (reviewed in Lew & Bookchin, 2005). Calcium uptake and the associated endocytotic process are both stimulated by deoxygenation-induced sickling (Murphy et al, 1987; Rhoda et al, 1990). We hypothesized that increased intracellular calcium in sickle erythrocytes may also cause a chronic energy deficit by simultaneously increasing the demand for ATP and activating AMPD. Furthermore, this Adenine Nucleotide metabolic dysregulation could impact on the clinical manifestations of this disorder. However, ATP concentrations are greater in reticulocyte-enriched RBC fractions (Brok et al, 1966; Cohen et al, 1976; Clark et al, 1978), indicating that the higher circulating levels of reticulocytes in SCD would confound the results of a comparative analysis of Adenine Nucleotide and IMP concentrations in RBCs isolated from these individuals versus normal subjects. The present study addressed this issue by using a combination of centrifugation and immunomagnetic separation techniques to reduce reticulocyte levels in RBC samples, which allowed for a more accurate comparison of Adenine Nucleotide and IMP levels in sickle and normal erythrocytes.
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amp deaminase activation contributes to accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in sickle cell erythrocytes
Blood, 2005Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of Adenine Nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte Adenine Nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the Adenine Nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP. Data in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p These combined results demonstrate AMPD3 enzyme activation and accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and Adenine supplementation, may have a therapeutic benefit in SCD.
Richard L Sabina - One of the best experts on this subject based on the ideXlab platform.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Ca2+-calmodulin (Ca2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate Adenine Nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total Adenine Nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0.05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca2+-CaM activates AMPD in SCD. Finally, Adenine treatment (100 micromol/l) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)-exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0.05 compared to untreated samples). We conclude that Ca2+-CaM activation of AMPD contributes to increased turnover of the Adenine Nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Erythrocytes are unique among human tissues and cells because they do not contain enough adenylosuccinate synthetase activity to sustain measurable anabolic flow through the inosine monophosphate (IMP) to adenosine monophosphate (AMP) branch point of purine Nucleotide biosynthesis (Bishop, 1960; Lowy & Dorfman, 1970). The immediate metabolic consequence of this non-functional IMP to AMP branch point pathway is an inability to synthesize Adenine Nucleotides from either the de novo pathway or from the salvage pathway for hypoxanthine, the most abundant purine compound in the circulation (see Fig S1). Although enzymes are present for the salvage synthesis of AMP directly from adenosine and Adenine, the circulating levels of both compounds are normally quite low, i.e., <1 µmol/1 (Ericson et al, 1980; Moser et al, 1989). Consequently, erythrocytes have a severely limited capacity for maintaining their Adenine Nucleotide pool and the associated cellular energy reserves. This is particularly evident under conditions of energy imbalance that result in a net turnover of ATP, which can lead to activation of catabolic enzymes and an accelerated loss of Adenine Nucleotides. AMP deaminase (AMPD) converts AMP to IMP, a catabolite that has no anabolic route back into the erythrocyte Adenine Nucleotide pool (Fig S1). Thus, it has long been recognized that erythrocytes attempt to preserve their Adenine Nucleotide pool by maintaining an intracellular environment that minimizes AMPD catalytic activity (Askari, 1966). Several small molecule inhibitors of erythrocyte AMPD have been identified, including inorganic phosphate (Askari, 1966, Lian & Harkness, 1974; Yun & Suelter, 1978), 2,3-diphosphoglycerate (Askari & Rao, 1968; Lian & Harkness, 1974; Yun & Suelter, 1978), and phosphatidylinositol 4,5-bisphosphate (Sims et al, 1999), with measured Ki values that are similar to estimated intracellular concentrations in erythrocytes (Bontemps et al, 1986). The inherent capacity of normal erythrocytes to maintain Adenine Nucleotides during an energy crisis is reflected by the accumulation of AMP, rather than IMP, during short periods of accelerated ATP turnover in response to glucose deprivation (Bontemps et al, 1986; Mahnke & Sabina, 2005). Conversely, IMP, rather than AMP, accumulates in calcium-permeabilized erythrocytes (Almarez et al, 1988; Engstrom et al, 1996). The underlying mechanism responsible for rapid IMP accumulation under these conditions was not immediately evident because calcium activation of AMPD was lost upon dilution of the haemolysate or after the enzyme was partially purified (Almarez & Garcia-Sancho, 1989). This confounding issue was subsequently clarified by the discovery that calcium-calmodulin (Ca2+-CaM) activates erythrocyte AMPD and overcomes the inhibitory mechanisms inherent to these cells (Mahnke & Sabina, 2005). Similar Adenine Nucleotide metabolic dysregulation exists in clinical disorders of disturbed erythrocyte calcium homeostasis. For example, familial phosphofructokinase deficiency (FPD) is accompanied by a mild compensated anaemia and erythrocytes isolated from these individuals contain increased intracellular calcium (Waldenstrom et al, 2001) and a smaller Adenine Nucleotide pool concomitant with elevated levels of IMP (Ronquist et al, 2001; Sabina et al, 2006). The slowing of additional IMP accumulation during an experimental period of energy imbalance when FPD erythrocytes were pre-incubated with a diffusible CaM antagonist (Sabina et al, 2006) provided evidence that the protein–protein interaction between erythrocyte AMPD and Ca2+-CaM contributes to this metabolic dysregulation. Intracellular calcium is also elevated in sickle erythrocytes (Eaton et al, 1973; Palek, 1977; Bookchin & Lew, 1980), where it is primarily sequestered into intracellular vesicles by an inside-out ATPase pump (reviewed in Lew & Bookchin, 2005). Calcium uptake and the associated endocytotic process are both stimulated by deoxygenation-induced sickling (Murphy et al, 1987; Rhoda et al, 1990). We hypothesized that increased intracellular calcium in sickle erythrocytes may also cause a chronic energy deficit by simultaneously increasing the demand for ATP and activating AMPD. Furthermore, this Adenine Nucleotide metabolic dysregulation could impact on the clinical manifestations of this disorder. However, ATP concentrations are greater in reticulocyte-enriched RBC fractions (Brok et al, 1966; Cohen et al, 1976; Clark et al, 1978), indicating that the higher circulating levels of reticulocytes in SCD would confound the results of a comparative analysis of Adenine Nucleotide and IMP concentrations in RBCs isolated from these individuals versus normal subjects. The present study addressed this issue by using a combination of centrifugation and immunomagnetic separation techniques to reduce reticulocyte levels in RBC samples, which allowed for a more accurate comparison of Adenine Nucleotide and IMP levels in sickle and normal erythrocytes.
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Adenine Nucleotide pool perturbation is a metabolic trigger for amp deaminase inhibitor based herbicide toxicity
Plant Physiology, 2007Co-Authors: Richard L Sabina, Annalisa Paul, Robert J Ferl, Bernd Laber, Stephen D LindellAbstract:AMP deaminase (AMPD) is essential for plant life, but the underlying mechanisms responsible for lethality caused by genetic and herbicide-based limitations in catalytic activity are unknown. Deaminoformycin (DF) is a synthetic modified nucleoside that is taken up by plant cells and 5′-phosphorylated into a potent transition state-type inhibitor of AMPD. Systemic exposure of Arabidopsis (Arabidopsis thaliana) seedlings to DF results in dose-dependent (150–450 nm) and time-dependent decreases in plant growth that are accompanied by 2- to 5-fold increases in the intracellular concentrations of all Adenine riboNucleotides. No measurable rescue is observed with either hypoxanthine or xanthine (250 μm), indicating that downstream effects of AMPD inhibition, such as limitations in Adenine-to-guanine Nucleotide conversion or ureide synthesis, do not play important roles in DF toxicity. However, Adenine (250 μm) acts synergistically with a nontoxic dose of DF (150 nm) to produce growth inhibition and Adenine Nucleotide pool expansion comparable to that observed with a toxic concentration of the herbicide alone (300 nm). Conversely, Adenine alone (60–250 μm) has no measurable effects on these parameters. These combined results support the hypothesis that AMPD is the primary intracellular target for this class of herbicides and strongly suggest that Adenine Nucleotide accumulation is a metabolic trigger for DF toxicity. AMP binds to 14-3-3 proteins and can interrupt client interactions that appear to drive their distributions. Trichome subcellular localization of the phi isoform is disrupted within 8 to 24 h after seedlings are semisubmersed in a solution of DF (100 nm), further suggesting that disrupted 14-3-3 protein function plays a role in the associated herbicidal activity.
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amp deaminase activation contributes to accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in sickle cell erythrocytes
Blood, 2005Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of Adenine Nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte Adenine Nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the Adenine Nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP. Data in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p These combined results demonstrate AMPD3 enzyme activation and accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and Adenine supplementation, may have a therapeutic benefit in SCD.
Nancy J Wandersee - One of the best experts on this subject based on the ideXlab platform.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Ca2+-calmodulin (Ca2+-CaM) activates erythrocyte adenosine monophosphate deaminase (AMPD) in conditions of disturbed calcium homeostasis, prompting us to investigate Adenine Nucleotide metabolic dysregulation in sickle cell disease (SCD). However, higher ATP concentrations in reticulocytes, compared to erythrocytes, confound a comparative evaluation of SCD and normal RBCs. Therefore, a combination of centrifugation and antiCD71-labelled magnetic bead selection was used to prepare reticulocyte-poor fractions (reticulocytes <4% of total RBCs) of SCD RBCs. ATP and total Adenine Nucleotide concentrations were 12% lower in sickle erythrocytes compared to normal erythrocytes and inosine monophosphate (IMP) concentrations were threefold elevated (all P < 0.05). Furthermore, preincubation with a diffusible CaM antagonist slowed IMP accumulation in sickle erythrocytes during an experimental period of energy imbalance, thus showing that Ca2+-CaM activates AMPD in SCD. Finally, Adenine treatment (100 micromol/l) of ex vivo SCD RBCs significantly expanded ATP levels (16% higher) and reduced phosphatidylserine (PS)-exposure, specifically those cells with the highest levels of PS externalization (46% fewer events) (both P-values <0.05 compared to untreated samples). We conclude that Ca2+-CaM activation of AMPD contributes to increased turnover of the Adenine Nucleotide pool in sickle erythrocytes and that this metabolic dysregulation promotes PS exposure that may contribute to the pathogenesis of SCD.
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ca2 cam activation of amp deaminase contributes to Adenine Nucleotide dysregulation and phosphatidylserine externalization in human sickle erythrocytes
British Journal of Haematology, 2009Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Erythrocytes are unique among human tissues and cells because they do not contain enough adenylosuccinate synthetase activity to sustain measurable anabolic flow through the inosine monophosphate (IMP) to adenosine monophosphate (AMP) branch point of purine Nucleotide biosynthesis (Bishop, 1960; Lowy & Dorfman, 1970). The immediate metabolic consequence of this non-functional IMP to AMP branch point pathway is an inability to synthesize Adenine Nucleotides from either the de novo pathway or from the salvage pathway for hypoxanthine, the most abundant purine compound in the circulation (see Fig S1). Although enzymes are present for the salvage synthesis of AMP directly from adenosine and Adenine, the circulating levels of both compounds are normally quite low, i.e., <1 µmol/1 (Ericson et al, 1980; Moser et al, 1989). Consequently, erythrocytes have a severely limited capacity for maintaining their Adenine Nucleotide pool and the associated cellular energy reserves. This is particularly evident under conditions of energy imbalance that result in a net turnover of ATP, which can lead to activation of catabolic enzymes and an accelerated loss of Adenine Nucleotides. AMP deaminase (AMPD) converts AMP to IMP, a catabolite that has no anabolic route back into the erythrocyte Adenine Nucleotide pool (Fig S1). Thus, it has long been recognized that erythrocytes attempt to preserve their Adenine Nucleotide pool by maintaining an intracellular environment that minimizes AMPD catalytic activity (Askari, 1966). Several small molecule inhibitors of erythrocyte AMPD have been identified, including inorganic phosphate (Askari, 1966, Lian & Harkness, 1974; Yun & Suelter, 1978), 2,3-diphosphoglycerate (Askari & Rao, 1968; Lian & Harkness, 1974; Yun & Suelter, 1978), and phosphatidylinositol 4,5-bisphosphate (Sims et al, 1999), with measured Ki values that are similar to estimated intracellular concentrations in erythrocytes (Bontemps et al, 1986). The inherent capacity of normal erythrocytes to maintain Adenine Nucleotides during an energy crisis is reflected by the accumulation of AMP, rather than IMP, during short periods of accelerated ATP turnover in response to glucose deprivation (Bontemps et al, 1986; Mahnke & Sabina, 2005). Conversely, IMP, rather than AMP, accumulates in calcium-permeabilized erythrocytes (Almarez et al, 1988; Engstrom et al, 1996). The underlying mechanism responsible for rapid IMP accumulation under these conditions was not immediately evident because calcium activation of AMPD was lost upon dilution of the haemolysate or after the enzyme was partially purified (Almarez & Garcia-Sancho, 1989). This confounding issue was subsequently clarified by the discovery that calcium-calmodulin (Ca2+-CaM) activates erythrocyte AMPD and overcomes the inhibitory mechanisms inherent to these cells (Mahnke & Sabina, 2005). Similar Adenine Nucleotide metabolic dysregulation exists in clinical disorders of disturbed erythrocyte calcium homeostasis. For example, familial phosphofructokinase deficiency (FPD) is accompanied by a mild compensated anaemia and erythrocytes isolated from these individuals contain increased intracellular calcium (Waldenstrom et al, 2001) and a smaller Adenine Nucleotide pool concomitant with elevated levels of IMP (Ronquist et al, 2001; Sabina et al, 2006). The slowing of additional IMP accumulation during an experimental period of energy imbalance when FPD erythrocytes were pre-incubated with a diffusible CaM antagonist (Sabina et al, 2006) provided evidence that the protein–protein interaction between erythrocyte AMPD and Ca2+-CaM contributes to this metabolic dysregulation. Intracellular calcium is also elevated in sickle erythrocytes (Eaton et al, 1973; Palek, 1977; Bookchin & Lew, 1980), where it is primarily sequestered into intracellular vesicles by an inside-out ATPase pump (reviewed in Lew & Bookchin, 2005). Calcium uptake and the associated endocytotic process are both stimulated by deoxygenation-induced sickling (Murphy et al, 1987; Rhoda et al, 1990). We hypothesized that increased intracellular calcium in sickle erythrocytes may also cause a chronic energy deficit by simultaneously increasing the demand for ATP and activating AMPD. Furthermore, this Adenine Nucleotide metabolic dysregulation could impact on the clinical manifestations of this disorder. However, ATP concentrations are greater in reticulocyte-enriched RBC fractions (Brok et al, 1966; Cohen et al, 1976; Clark et al, 1978), indicating that the higher circulating levels of reticulocytes in SCD would confound the results of a comparative analysis of Adenine Nucleotide and IMP concentrations in RBCs isolated from these individuals versus normal subjects. The present study addressed this issue by using a combination of centrifugation and immunomagnetic separation techniques to reduce reticulocyte levels in RBC samples, which allowed for a more accurate comparison of Adenine Nucleotide and IMP levels in sickle and normal erythrocytes.
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amp deaminase activation contributes to accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in sickle cell erythrocytes
Blood, 2005Co-Authors: Richard L Sabina, Nancy J Wandersee, Cheryl A HilleryAbstract:Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of Adenine Nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte Adenine Nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the Adenine Nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP. Data in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p These combined results demonstrate AMPD3 enzyme activation and accelerated Adenine Nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and Adenine supplementation, may have a therapeutic benefit in SCD.
Jolita Ciapaite - One of the best experts on this subject based on the ideXlab platform.
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modular kinetic analysis of the Adenine Nucleotide translocator mediated effects of palmitoyl coa on the oxidative phosphorylation in isolated rat liver mitochondria
Diabetes, 2005Co-Authors: Jolita Ciapaite, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Marijke J. WagnerAbstract:To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial Adenine Nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Δψ) and the fraction of matrix ATP as intermediates. We found that 5 μmol/l palmitoyl-CoA inhibited Adenine Nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial “work” flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Δψ increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Δψ increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Δψ and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on Adenine Nucleotide translocator.
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application of modular control analysis to inhibition of the Adenine Nucleotide translocator by palmitoyl coa
Molecular Biology Reports, 2002Co-Authors: Jolita Ciapaite, Gerco Van EikenhorstAbstract:Modular kinetic analysis was used to characterize inhibition of Adenine Nucleotide translocation by palmitoyl-CoA in isolated rat-liver mitochondria. To this purpose, oxidative phosphorylation has been divided into two modules with the fraction of matrix ATP as linking intermediate. The Adenine Nucleotide translocator is the matrix ATP-consuming module and the remainder of oxidative phosphorylation (ATP synthesis, respiratory chain and transport of phosphates and respiratory substrate) is the matrix ATP-producing module. We found that palmitoyl-CoA inhibits ATP-consuming module (ANT) and has no effect on ATP-producing module. There were no significant differences between kinetic curves obtained with oligomycin and myxothiazol, inhibitors that have opposite effect on membrane potential, suggesting that the use of the fraction of matrix ATP as the only intermediate is a good approximation. A new method has been used to determine the fraction of ATP in the mitochondrial matrix.
Gerco Van Eikenhorst - One of the best experts on this subject based on the ideXlab platform.
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modular kinetic analysis of the Adenine Nucleotide translocator mediated effects of palmitoyl coa on the oxidative phosphorylation in isolated rat liver mitochondria
Diabetes, 2005Co-Authors: Jolita Ciapaite, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Marijke J. WagnerAbstract:To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial Adenine Nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Δψ) and the fraction of matrix ATP as intermediates. We found that 5 μmol/l palmitoyl-CoA inhibited Adenine Nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial “work” flux was allowed to vary, palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Δψ increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Δψ increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Δψ and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on Adenine Nucleotide translocator.
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application of modular control analysis to inhibition of the Adenine Nucleotide translocator by palmitoyl coa
Molecular Biology Reports, 2002Co-Authors: Jolita Ciapaite, Gerco Van EikenhorstAbstract:Modular kinetic analysis was used to characterize inhibition of Adenine Nucleotide translocation by palmitoyl-CoA in isolated rat-liver mitochondria. To this purpose, oxidative phosphorylation has been divided into two modules with the fraction of matrix ATP as linking intermediate. The Adenine Nucleotide translocator is the matrix ATP-consuming module and the remainder of oxidative phosphorylation (ATP synthesis, respiratory chain and transport of phosphates and respiratory substrate) is the matrix ATP-producing module. We found that palmitoyl-CoA inhibits ATP-consuming module (ANT) and has no effect on ATP-producing module. There were no significant differences between kinetic curves obtained with oligomycin and myxothiazol, inhibitors that have opposite effect on membrane potential, suggesting that the use of the fraction of matrix ATP as the only intermediate is a good approximation. A new method has been used to determine the fraction of ATP in the mitochondrial matrix.