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

Maren R Laughlin,David Thompson

doi:10.1074/jbc.271.46.28977

Maren R Laughlin, David Thompson

Open Access

https://doi.org/10.1074/jbc.271.46.28977

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Abstract

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 approximately 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.

Highlights

The divalent cationophore A23187 was used to change the concentration of intracellular Mg2ϩ in human erythrocytes, which are otherwise impermeable to magnesium
Free Magnesium—Fig. 1 shows 10-min 31P NMR spectra of a 50% suspension of washed human erythrocytes taken before and after the addition of A23187 in the presence of 1 mM EGTA and without added Mg2ϩ. [Mg2ϩ]i was determined from ⌬ATP␣␤ in 31P NMR spectra by the method of Gupta et al (4), which accounts for the selective oxygen-dependent binding of 2,3-DPG and ATP to hemoglobin
While [Mg2ϩ]i decreases in every case, [Mg2ϩ]e appears to be buffered by the erythrocyte and changes in such a way as to approach 0.32 mM

Summary

EXPERIMENTAL PROCEDURES

Erythrocyte Preparation—7 ml of venous blood was taken from healthy volunteers who had given informed consent, under the guidelines of the George Washington University Medical Center IRB. Neutralized extracts were lyophilized and dissolved in 0.4 ml of 2H2O, and fully relaxed proton-decoupled 13C NMR spectra were acquired (60° pulses, 10-s delays, CPD bilevel decoupling) for determination of the C-3/C-2 [13C]lactate ratio used to calculate the pentose phosphate shunt activity. Intracellular Mg2ϩ and MgATP Determination—[Mg2ϩ]i and [MgATP] were calculated from the chemical shift difference between ␣and ␤-ATP, and total 2,3-DPG and ATP were measured in 31P NMR. 1. 10-min 31P NMR spectra of a 50% suspension of washed human erythrocytes taken at 121 MHz. The lower spectrum shows cells incubated with 10 mM glucose and 1 mM EGTA and without added Mg2ϩ. Glucose Utilization—␣- and ␤-[2-13C]glucose resonance areas were combined, and the results were normalized by setting peaks in the first 13C NMR spectra (t ϭ 0 –10 min) equal to the total solution [glucose] measured at t ϭ 0.

Glc 3 3 CO2 ϩ 2Fru-6-P ϩ 1 GAP 3 3 CO2 ϩ 5 lactate3 Ϫ 13Clactate

RESULTS

DISCUSSION