The regulatory role for magnesium in glycolytic flux of the human erythrocyte.
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.
Department of Surgery, George Washington University Medical Center, Washington, D. C. 20037, USA.
M R
MR
Laughlin
D
D
Thompson
D001731:Bisphosphoglycerate Mutase / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D006593:Hexokinase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010427:Pentose Phosphate Pathway; D010732:Phosphofructokinase-1 / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism
D000001:Calcimycin; D006593:Hexokinase; D010732:Phosphofructokinase-1; D010735:Phosphoglycerate Kinase; D001731:Bisphosphoglycerate Mutase; D008274:Magnesium; D005947:Glucose