Direct determination of 2,3-diphosphoglycerate

Rose, Zelda B.; Liebowitz, Judith

doi:10.1016/0003-2697(70)90023-0

Cited by 243 publications

(58 citation statements)

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“…3) decreases from 384 525 pM at pH,=7.41 to 286k20pM at pH 7.04. The K, values differ from those reported on purified preparations by Harnkness and Roth [12] and by Rose and Liebowitz [13] but agrees with that, found by Chiba and Sasaki [24]. Since in the presence of glucose below pH 6.9 2,3-P,glycerate breaks down at the same rate as without glucose, the 2,3-P,glycerate mutase must be practically completely inhibited.…”

Section: Discussionsupporting

confidence: 83%

“…2), suggests a cooperative behaviour of the enzyme. The strong effect of pH on the activity of the 2,3-P,glycerate mutase has not been observed so far on purified enzyme preparations [13]. The time required for the attainment of a new steady state of 2,3-P,glycerate, when the rate of formation of 2,3-P,glycerate equals its breakdown of about 18 h, i.r.…”

Section: Discussionmentioning

confidence: 95%

“…For the determination of 2,3-P,glycerate two procedures were applied. For the range from 5 to 0.5 mM the method of Rose and Liebowitz [8] was used. The inorganic phosphate, formed stoichiometrically from 2,3-P,glycerate was determined according to the method of Taussky and Shorr [9].…”

Section: Determination Of Metabolitesmentioning

confidence: 99%

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pH‐Dependent Changes of 2,3‐Bisphosphoglycerate in Human Red Cells during Transitional and Steady States in vitro

Rapoport

Berger

Elsner

et al. 1977

European Journal of Biochemistry

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A systematic study of the pH-dependent changes in the range 6.6 -7.4 of 2,3-bisphosphoglycerate and the adenine nucleotides was performed in the presence and absence of glucose during transitional and steady states.1. The results indicate that 2,3-bisphosphoglycerate phosphatase breaks down 2,3-bisphosphoglycerate nearly independent of pH at a rate of 480 pmol 2,3-bisphosphoglycerate x 1 cells-' x h-'. 2,3-Bisphosphoglycerate mutase is practically completely inhibited below pH 6.9. An increased K, 2,3-bisphosphoglycerate is postulated. The 2,3-bisphosphoglycerate level in the presence of glucose reaches a pH-dependent steady state after about 18 h in keeping with theoretical predictions.2. The constancy of the adenine nucleotides in the presence of glucose regardless of the rate of glycolysis indicates a close coupling between ATP formation and breakdown. The decline of the sum of adenine nucleotides appears to be determined by the rate of AMP breakdown.3. The share of the 2,3-bisphosphoglycerate bypass in the steady state decreases from 24% at pH 7.4 to 12 % at pH 7.04. The initially decreased glycolysis at low pH values increases in long-term experiments with lower 2,3-bisphosphoglycerate levels. The formation of pyruvate corresponds to the breakdown of 2,3-bisphosphoglycerate after consumption of an unknown reducing substance.The original observations of a breakdown of 2,3-P,glycerate [l -31 at low pH values, both in uivo and in vitro have been confirmed repeatedly. Nevertheless a systematic study of this phenomenon has not been performed so far, nor has its mechanism been elucidated. At present there is a lack of understanding by what mechanism steady state concentrations of 2,3-P,-glycerate are established under various physiological and pathological conditions. The experiments carried out so far in vitro do not permit valid extrapolations to the conditions in vivo. The purpose of the present study was an investigation of the pH-dependent kinetics of the changes of 2,3-P2glycerate both during transitional and steady states. It included an assessment of both the over-all flux through glycolysis as well as through the 2,3-P2glycerate bypass. In addition the behaviour of the adenine nucleotides was studied.Abbreviations. 2,3-P2glycerate, 2,3-bisphosphoglycerate; 1,3-Enzymes.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 95%

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pH‐Dependent Changes of 2,3‐Bisphosphoglycerate in Human Red Cells during Transitional and Steady States in vitro

Rapoport

Berger

Elsner

et al. 1977

European Journal of Biochemistry

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“…DPG-which affects erythrocyte capacity to release oxygen in tissue and generally increases when HB decreases-and ATP in erythrocytes were measured spectrophotometrically. 18,19 Determination of Osmotic Resistance. One hundred milliliters of packed erythrocytes were carefully added to 13 tubes containing 1 mL each of 0.85%-0% saline in distilled water.…”

Section: Methodsmentioning

confidence: 99%

Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C

et al. 2004

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Hemolysis is a frequent adverse effect of ribavirin (RBV). It has been suggested that oxidative stress plays a role, but mechanisms and predictive risk factors for severe forms remain unknown. Markers of redox status were determined in erythrocytes of 34 patients with hepatitis C-four of them with glucose-6-phosphate-dehydrogenase (G6PD) deficiencybefore and during treatment with RBV and interferon (IFN) and were compared with 10 healthy control subjects. In addition, erythrocytes were incubated with RBV, and the effects of dipyridamole (DPD), diethylmaleate (DEM), and glutathione ester (GSHE) were studied in vitro. Of the 30 patients without G6PD deficiency who were treated with RBV and IFN-␣, five developed major hemolysis (⌬ hemoglobin > 6 g/dL) and 25 developed minor hemolysis (⌬ hemoglobin < 2.5 g/dL). Patients with major hemolysis had lower median pretreatment values of membrane protein sulfhydrils than patients with minor hemolysis (28.4 vs. 36.7 nmol/mg, P < .001). Erythrocytes of G6PD-deficient patients were not more susceptible to RBV-induced hemolysis. In in vitro incubations of erythrocytes, DEM enhanced the RBV-induced decrease of glutathione, protein sulfhydrils, and osmotic resistance. Supplementation of GSHE and DPD prevented the RBV-induced decrease in osmotic resistance, adenosyl triphosphate (ATP), and 2,3-diphosphoglycerate (DPG), the loss of glutathione and protein sulfhydrils, and the formation of thiobarbituric acid reactive substances (TBARs). In conclusion, the data indicate that low membrane protein sulfhydrils prior to therapy but not G6PD deficiency are predictive of RBV-induced major hemolysis. In vitro, GSHE and DPD reduce the RBV-associated oxidative stress in erythrocytes and prevent the increase in osmotic fragility, suggesting that these compounds might decrease the risk of hemolysis in patients. (HEPATOLOGY 2004;39:1248 -1255 R ibavirin (RBV) is a synthetic nucleoside analogue used in the treatment of chronic hepatitis C in combination with interferon (IFN)-␣. The addition of RBV has increased the probability of sustained response to approximately 40% if combined with conventional IFN 1,2 and to over 50% if combined with pegylated IFN. 3 RBV actively enters cells by way of the es-nucleoside membrane transporter 4 and subsequently undergoes phosphorylation at the expense of adenosyl triphosphate (ATP). 5,6 In contrast to nucleated cells such as hepatocytes, phosphorylation is irreversible in the anucleate erythrocytes, and phosphorylated RBV accumulates because it cannot be exported by the nucleoside carrier. Therefore, the concentration of RBV in erythrocytes is 50-to 70-fold higher than the therapeutic plasma concentration of 9 -15 M. 5,7,8 The high concentration of RBV in erythrocytes is probably responsible for the hemolysis occurring during RBV therapy. A decrease in hemoglobin (HB) is frequently observed in patients who receive RBV,

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“…The hydrolysis of 2,3-DPG is physiologically stimulated by 2-phosphoglycolate, a normal constituent of red blood cells (4). BPGM also displays a mutase reaction similar to that of the glycolytic enzyme monophosphoglycerate mutase (MPGM, EC 5.4.2.1) which reversibly converts glycerate 3-phosphate (3-PG) to glycerate 2-phosphate (5,6).…”

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confidence: 99%

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

Ravel

Craescu

Arous

et al. 1997

Journal of Biological Chemistry

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The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys 22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the K a value of the activator, whereas it caused no change in other catalytic activities or in the K m values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys 22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding.Kinetic experiments showed that the K a of the cofactor and the K m of 3-PG were affected by the substitution of Ser 23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified K m value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg 89 is specifically involved in monophosphoglycerates binding.CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species.In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys 22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.

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Direct determination of 2,3-diphosphoglycerate

Cited by 243 publications

References 4 publications

pH‐Dependent Changes of 2,3‐Bisphosphoglycerate in Human Red Cells during Transitional and Steady States in vitro

pH‐Dependent Changes of 2,3‐Bisphosphoglycerate in Human Red Cells during Transitional and Steady States in vitro

Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C

Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site

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