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The Regulatory Role for Magnesium in Glycolytic Flux of the Human Erythrocyte*

  • Maren R. Laughlin
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Surgery, George Washington University Medical Center, Washington, D. C. 20037
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  • David Thompson
    Affiliations
    Department of Surgery, George Washington University Medical Center, Washington, D. C. 20037
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 15, 1996DOI:https://doi.org/10.1074/jbc.271.46.28977
      31P NMR was used to measure the intracellular free magnesium concentration ([Mg2+]i) in human erythrocytes while [Mg2+]i was changed between 0.01 and 1.2 mM using the divalent cationophore A23187. 13C NMR and [2-13C]glucose were used to determine the kinetic effects of [Mg2+]i by measuring the flux through several parts of the glucose pathway. Glucose utilization was strongly dependent on [Mg2+]i, with half-maximal flux occurring at 0.03 mM. The rate-limiting step was most likely at phosphofructokinase, which has a Km(Mg2+) of 0.025 mM in the purified enzyme. Phosphorylated glycolytic intermediate concentration was also strongly dependent on [Mg2+]i and [MgATP], and glucose transport plus hexokinase may have been partially rate-determining at [Mg2+]i below ∼0.1 mM. The pentose phosphate shunt activity was too low to determine the dependence on [Mg2+]i. Phosphoglycerate kinase and 2,3-diphosphoglycerate mutase fluxes were also measured, but were not rate-limiting for glycolysis and showed no Mg2+ dependence. Human erythrocyte [Mg2+]i varies between 0.2 mM (oxygenated) and 0.6 mM (deoxygenated), well above the measured [Mg2+]i(1/2). It is unlikely, then, that [Mg2+]i plays a regulatory role in normal erythrocyte glycolysis.

      INTRODUCTION

      Many of the enzymes in the metabolic pathways that utilize glucose have a requirement for magnesium as demonstrated in kinetic studies of isolated enzymes (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ,
      • Gerber G.
      • Preissler H.
      • Heinrich R.
      • Rapoport S.M.
      ,
      • Otto M.
      • Heinrich R.
      • Kuhn B.
      • Jacobasch G.
      ). The Km values for Mg2+ in the glycolytic enzymes of the human erythrocyte are between 1 and 2.3 mM for hexokinase (maximum activity at 37°C = 11 μmol/h/ml of erythrocytes), 0.025 mM for phosphofructokinase (PFK)
      The abbreviations used are: PFK
      phosphofructokinase
      PGK
      phosphoglycerate kinase
      [Mg2+]i
      intracellular free magnesium concentration
      [Mg2+]e
      extracellular free magnesium concentration; 2,3-DPG, 2,3-diphosphoglycerate.
      (200 μmol/h/ml), 0.3 mM for phosphoglycerate kinase (PGK) (3000 μmol/h/ml), and 1 mM for pyruvate kinase (230 μmol/h/ml) (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ,
      • Otto M.
      • Heinrich R.
      • Kuhn B.
      • Jacobasch G.
      ). [Mg2+]i in oxygenated erythrocytes is 0.2 mM, which rises to 0.6 mM in the absence of oxygen due to the oxygen-dependent behavior of ATP binding to hemoglobin (
      • Gupta R.K.
      • Benovic J.L.
      • Rose Z.B.
      ). Glucose utilization is concurrently increased by ∼23-33% in the deoxygenated cell (
      • Murphy J.R.
      ,
      • Schrader M.C.
      • Eskey C.J.
      • Simplaceanu V.
      • Ho C.
      ). Since [Mg2+]i is near the measured Km for three of the potentially rate-limiting kinases and because both [Mg2+]i and the glycolytic rate are modulated as oxygen tension changes, it stands to reason that [Mg2+]i is important in erythrocyte glycolysis.
      The divalent cationophore A23187 was used to change the concentration of intracellular Mg2+ in human erythrocytes, which are otherwise impermeable to magnesium. The distribution of Mg2+ across the cell membrane is then a function of membrane potential, Vm (Equation 1) (
      • Flatman P.
      • Lew V.L.
      ).
      Mg2+i=exp2FVm/RTMg2+e
      (Eq. 1)


      Intracellular Mg2+ was measured from the chemical shift of the 31P NMR signals of the α- and β-phosphate groups of ATP (
      • Gupta R.K.
      • Benovic J.L.
      • Rose Z.B.
      ). 13C NMR was used to measure [2-13C]glucose utilization and to estimate the flux through several of the enzyme systems in the glycolytic pathway at [Mg2+]e between 0.01 and 1.00 mM: total glucose utilization, PFK flux, 2,3-DPG turnover, PGK flux, and pentose phosphate pathway flux (
      • Schrader M.C.
      • Eskey C.J.
      • Simplaceanu V.
      • Ho C.
      ).

      DISCUSSION

      Erythrocyte glycolysis depends on [Mg2+]i, with an overall [Mg2+]i(1/2) of ∼0.03 mM, which is an order of magnitude below physiologically important levels. In the normal oxygen delivery cycle, [Mg2+]i varies between 0.2 mM (oxygenated) and 0.6 mM (deoxygenated) (
      • Gupta R.K.
      • Benovic J.L.
      • Rose Z.B.
      ), but at constant oxygen tension, appears to be well buffered. Erythrocytes contain ∼3.5 mmol of total magnesium/kg of water and three to four distinct pools of buffering molecules: 100 μM buffer with Km≈ 0.03 μM, 2 mM buffer with Km≈ 25-50 μM, and ∼20-30 mM buffer with Km≈ 1-4 mM (
      • Flatman P.
      • Lew V.L.
      ). Under certain pathological conditions, human erythrocyte [Mg2+]i can decrease, but falls to only 0.13 ± 0.02 mM in renal magnesium loss (
      • Geven W.B.
      • Vogels-Mentink W.B.
      • Willems J.L.
      • Os C.H. v.
      • Hilbers C.W.
      • Joordens J.J.M.
      • Rijksen G.
      • Monens L.A.H.
      ) or to 0.16 mM after 3 weeks of magnesium deficiency (
      • Rude R.K.
      • Stephen A.
      • Nadler J.
      ).
      Calculated [MgATP] correlates well with PFK flux (Fig. 8) and NMR-visible phosphomonoesters (Fig. 10). MgATP concentration is ∼0.48 mM at [Mg2+]i(1/2) = 0.03 mM in oxygenated cells. In the normal course of oxygenation/deoxygenation, it varies between 1.0 and 0.8 mM (
      • Gupta R.K.
      • Benovic J.L.
      • Rose Z.B.
      ), which may be in the regulatory range.
      Integration of the phosphomonoester region indicates that the phosphorylated intermediates including such compounds as Glc-6-P (4.7 ppm) and DHAP (4.37 ppm) increase during the incubation with glucose at high [Mg2+]i and decrease at low [Mg2+]i. The crossover point is ∼0.1 mM Mg2+ or ∼0.5 mM MgATP. Even though glucose utilization is very slow at low [Mg2+]i, the concentration of phosphorylated glycolytic intermediates falls, indicating that transport and/or phosphorylation of glucose (these steps cannot be distinguished in the present experiment) is lagging behind other potentially rate-limiting steps. At higher [Mg2+]i, the rate-limiting step must be later in the glycolytic pathway since phosphorylated intermediate pools build up. This implies that the combination of transport and phosphorylation of glucose is at least partially rate-limiting for glycolysis at low [Mg2+]i. The Ka(Mg2+) for purified hexokinase from human erythrocytes is between 1.0 and 2.3 mM, with a Mg2+ dependence observed up to 4 mM, and the Km(MgATP) is between 1 and 2 mM (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ,
      • Gerber G.
      • Preissler H.
      • Heinrich R.
      • Rapoport S.M.
      ). Since at even the highest [Mg2+]i studied the change in phosphomonoesters had not reached a maximum, our results in the intact cell are consistent with this rather high Ka(Mg2+) for hexokinase (Fig. 10). This activation of the early steps in glycolysis may be important for increasing glycolytic intermediates in deoxygenated cells, which have much higher [Mg2+]i and higher glycolytic rates.
      The 13C NMR experiment does not clearly indicate which step in the glycolytic pathway is rate-limiting. It may in fact change throughout the experiment; lactate accumulation changes the intracellular NADH/NAD+ ratio and inhibits glyceraldehyde-3-phosphate dehydrogenase (
      • Tilton W.M.
      • Seaman C.
      • Carriero D.
      • Piomelli S.
      ). On the other hand, decreases in pH have the greatest effect on PFK (
      • Minakami S.
      • Yoshikawa H.
      ). The present experiments do demonstrate a distinct Mg2+ dependence of glycolysis, and PFK has a clear dependence on Mg2+ and MgATP, while glyceraldehyde-3-phosphate dehydrogenase does not. The Km(Mg2+) for purified human erythrocyte PFK has been reported to be 0.025 mM (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ). In an analysis of the kinetics of PFK from rat erythrocytes, it appeared that Mg2+ in itself did not directly activate the enzyme (
      • Otto M.
      • Heinrich R.
      • Kuhn B.
      • Jacobasch G.
      ). It instead served three distinct roles: as part of the substrate MgATP (Km(MgATP) = 0.07 mM), to release inhibition by uncomplexed ATP (Ki(ATP) = 0.01 mM), and to inhibit PFK (Ki(Mg2+) = 0.44 mM). In the present study, there was no apparent decline in PFK flux at [Mg2+]i near or above the reported Ki, and the MgATP and ATP concentrations at half-maximal velocity were on the order of 0.4 and 0.6 mM, respectively, well above the reported activation and inhibition coefficients. Our results do not support this second model of PFK regulation. However, because of the similarity of our measured [Mg2+]i(1/2) to the reported Km for the isolated human enzyme, PFK does appear to be the primary rate-determining enzyme under our experimental conditions (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ).
      The pentose phosphate pathway and PGK flux were too low to solidly define their Mg2+ dependence in the present experiments. It is interesting that unlike other cells that do not have the 2,3-DPG shunt, low activity of PGK does not limit glycolytic flux in the erythrocyte. Purified PGK has a strong dependence on Mg2+, with a reported Km(Mg2+) of 0.3 mM and Km(MgATP) = 0.44 mM (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ). Our data show no rate dependence around these points, indicating that PGK flux is being limited by something else. Since PGK is the point at which net ATP production is regulated, perhaps its flux is limited by low ATP utilization in these experiments. PGK flux is more or less linear with PFK flux (Fig. 9), which implies that the concentration of the substrate 1,3-diphosphoglycerate is important.
      In many studies, including one done with 13C NMR, the time-averaged pentose phosphate shunt activity was significant, ∼17% of total glucose utilization in oxygenated human erythrocytes (
      • Murphy J.R.
      ,
      • Schrader M.C.
      • Eskey C.J.
      • Simplaceanu V.
      • Ho C.
      ,
      • Palsson B.O.
      • Narang A.
      • Joshi A.
      ), whereas in the present study, it was at most 4%. This may be due to differences in the experimental design. The previous experiments were carried out for very long periods of time, and it appears from the time courses of [2-13C]lactate and [3-13C]lactate that the pentose phosphate shunt activity was increased at the later times, when 2,3-DPG was falling and Pi was probably rising. It was important to keep 2,3-DPG, total ATP, and Pi constant in these experiments because all three regulate glycolysis. 2,3-DPG inhibits PFK, hexokinase, PGK, and pyruvate kinase. PGK and pyruvate kinase may be inhibited by ∼80% at the normal 2,3-DPG concentrations (
      • Ponce J.
      • Roth S.
      • Harkness D.R.
      ). Pi inhibits PFK and strongly stimulates hexokinase (
      • Minakami S.
      • Yoshikawa H.
      ). It is possible that as high energy phosphates fall and Pi increases, pentose phosphate shunt activity is increased.
      A second explanation for the low pentose phosphate shunt activity is the low rate of Ca2+ pumping that takes place in the presence of 1 mM EGTA. When A23187 and even the minute calcium concentrations found in deionized water are present simultaneously, erythrocyte heat and lactate production are both increased, and ATP falls precipitously due to a very active ATP-dependent Ca2+-H+ exchanger in the membrane (
      • Engstrom I.
      • Waldenstrom A.
      • Nilsson-Ehle P.
      • Ronquist G.
      ,
      • Ferreira H.G.
      • Lew V.L.
      ,
      • Engstrom I.
      • Waldenstrom A.
      • Ronquist G.
      ). Normal erythrocyte intracellular Ca2+ is maintained at vanishingly low levels by this Ca2+ pump and an impermeable cell membrane in the face of ∼1.2 mM ion in the plasma. The basal level of the Ca2+-ATPases most likely causes some turnover of nucleotides and associated pentose phosphate pathway activity, which would be absent in the present experiments (
      • Palsson B.O.
      • Narang A.
      • Joshi A.
      ).
      The slope of the intracellular versus extracellular free magnesium concentration is a measure of the square of the Donnan potential (r), which is a function of membrane potential, and is highly dependent on pH, ion concentrations, and cell volume (
      • Flatman P.
      • Lew V.L.
      ). The Donnan potential has apparently changed during the experiment from 0.90 to 0.76. If true, this would largely explain the tight correlation between the change in [Mg2+]i and its initial concentration (r2 = 0.983). However, [Mg2+]e also changes, but in such a way as to approach the concentration 0.32 mM. This implies a large extracellular buffer for magnesium with a KD in the range of its normal plasma concentration (0.5 mM). The existence of such a buffer has been noted in the literature (
      • Tongyhai S.
      • Rayssiguier Y.
      • Motta C.
      • Gueux E.
      • Maurois P.
      • Heaton F.W.
      ) and may consist of membrane-bound phospholipids. We hoped to fill all magnesium sites in the cells by a long preincubation at the experimental magnesium ion and ionophore concentrations. The changes in [Mg2+]e may therefore indicate a shift in the binding site concentration or dissociation constant. Clearly, it cannot be assumed that either [Mg2+]e or [Mg2+]i is constant when experiments with A23187 are conducted for long periods of time at high hematocrits.
      The ionophore seems to increase glycolysis; in our studies, it went up ∼100% from 2.1 × 10−4 in controls (data not shown) to 4 × 10−4 mmol/min/g of hemoglobin. This is similar to the results of Engstrom et al. (
      • Engstrom I.
      • Waldenstrom A.
      • Nilsson-Ehle P.
      • Ronquist G.
      ,
      • Ferreira H.G.
      • Lew V.L.
      ), who found an increase in glycolysis from 6.7 × 10−5 to 1.3 × 10−4 mmol/min/g of hemoglobin in the presence of A23187 and 3 mM Mg2+. It was also noted that the rates of glucose and [2-13C]glucose utilization are always greater than production of total lactate or labeled trioses (see Fig. 3 and Table I). This is not a new finding (
      • Schrader M.C.
      • Eskey C.J.
      • Simplaceanu V.
      • Ho C.
      ,
      • Tilton W.M.
      • Seaman C.
      • Carriero D.
      • Piomelli S.
      ). Since there is no apparent large increase in [2-13C]pyruvate or other labeled intermediates, it probably indicates slow lactate transport across the membrane or binding of lactate to cellular components accompanied by a decrease in NMR visibility of the bound fraction.
      In summary, [Mg2+]i(1/2) has been determined for glycolysis in the human erythrocyte and found to be 0.03 mM. The rate-limiting site is most likely to be PFK. Pentose phosphate shunt activity was too low to explore the magnesium dependence under these experimental conditions. 2,3-DPG mutase and PGK flux were not rate-limiting and therefore showed no Mg2+ dependence. Glucose transport and phosphorylation, as determined by concentration of and changes in total phosphomonoester compounds, have a strong dependence on [Mg2+]i and [MgATP]. These results indicate that there is a strong regulatory role for [Mg2+]i in the glycolytic pathways of the erythrocyte, but that [Mg2+]i(1/2) is far lower than the normal range of [Mg2+]i in the cell.

      Acknowledgments

      We thank Michael Salem, Anita Phillip, and Steven Malekzaleh for contributions to this work.

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