Mota de Freitas D, Amari L, Srinivasan C, Rong Q, Ramasamy R, Abraha A, Geraldes C F, Boyd M K
Department of Chemistry, Loyola University of Chicago, Illinois 60626.
Biochemistry. 1994 Apr 12;33(14):4101-10. doi: 10.1021/bi00180a002.
We investigated the mechanism of competition between Li+ and Mg2+ in Li(+)-loaded human red blood cells (RBCs) by making 7Li and 31P NMR and fluorescence measurements. We used 7Li NMR relaxation times to probe Li+ binding to the human RBC membrane and ATP; an increase in Mg2+ concentration caused an increase in both 7Li T1 and T2 values in packed Li(+)-loaded RBCs, in suspensions of Li(+)-loaded RBC ghosts, in suspensions of Li(+)-containing RBC membrane, and in aqueous solutions of ATP, indicating competition between Li+ and Mg2+ for binding sites in the membrane and ATP. We found that increasing concentrations of either Li+ or Mg2+ in the presence of human RBC membrane caused an increase in the 31P NMR chemical shift anisotropy parameter, which describes the observed axially symmetric powder pattern, indicating metal ion binding to the phosphate groups in the membrane. Competition between Li+ and Mg2+ for phosphate groups in ATP and in the RBC membrane was also observed by both fluorescence measurements and 31P NMR spectroscopy at low temperature. The ratio of the stoichiometric binding constants of Mg2+ to Li+ to the RBC membrane was approximately 20; the ratio of the conditional binding constants in the presence of a free intracellular ATP concentration of 0.2 mM was approximately 4, indicating that Li+ competes for approximately 20% of the Mg(2+)-binding sites in the RBC membrane. Our results indicate that, regardless of the spectroscopic method used, Li+ competes with Mg2+ for phosphate groups in both ATP and the RBC membrane; the extent of metal ion competition for the phosphate head groups of the phospholipids in the RBC membrane is enhanced by the presence of ATP. Competition between Li+ and Mg2+ for anionic phospholipids or Mg(2+)-activated proteins present in cell membranes may constitute the basis of a general molecular mechanism for Li+ action in human tissues.
我们通过进行7Li和31P核磁共振（NMR）以及荧光测量，研究了锂负载的人红细胞（RBC）中Li+与Mg2+之间的竞争机制。我们使用7Li NMR弛豫时间来探测Li+与人RBC膜和ATP的结合；Mg2+浓度的增加导致在 packed Li(+)-loaded RBCs、Li(+)-loaded RBC膜泡悬浮液、含Li(+)的RBC膜悬浮液以及ATP水溶液中，7Li T1和T2值均增加，这表明Li+与Mg2+在膜和ATP中的结合位点存在竞争。我们发现，在人RBC膜存在的情况下，Li+或Mg2+浓度的增加会导致31P NMR化学位移各向异性参数增加，该参数描述了观察到的轴对称粉末图谱，表明金属离子与膜中的磷酸基团结合。通过荧光测量和低温下的31P NMR光谱，也观察到了Li+与Mg2+在ATP和RBC膜中磷酸基团上的竞争。Mg2+与Li+与人RBC膜的化学计量结合常数之比约为20；在细胞内游离ATP浓度为0.2 mM时，条件结合常数之比约为4，这表明Li+竞争RBC膜中约20%的Mg(2+)-结合位点。我们的结果表明，无论使用何种光谱方法，Li+都与Mg2+在ATP和RBC膜中竞争磷酸基团；ATP的存在增强了金属离子对RBC膜中磷脂磷酸头部基团的竞争程度。Li+与Mg2+在细胞膜中存在的阴离子磷脂或Mg(2+)-激活蛋白之间的竞争，可能构成了Li+在人体组织中作用的一般分子机制的基础。
