Chung L, Kaloyanides G, McDaniel R, McLaughlin A, McLaughlin S
Biochemistry. 1985 Jan 15;24(2):442-52. doi: 10.1021/bi00323a030.
We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.
我们测量了多层磷脂囊泡的电泳迁移率、超声处理后的囊泡和多层囊泡的³¹P NMR谱，以及平面双层膜的电导率，以研究精胺和庆大霉素与膜的结合。精胺和庆大霉素与两性离子脂质磷脂酰胆碱的结合并不显著。我们测量了能使由磷脂酰胆碱与磷脂酰丝氨酸或磷脂酰肌醇的混合物形成的囊泡电荷反转的庆大霉素和精胺的浓度。通过这些测量，我们确定这些阳离子与这些带负电脂质的固有缔合常数均约为10 M⁻¹。该值比其他研究小组在文献中报道的表观结合常数低几个数量级，因为先前的研究没有明确考虑膜的负静电表面电位以及这些阳离子在膜相邻水相扩散双层中的积累。我们的主要结论是，如果带负电的磷脂占膜的比例小于50%，则 Gouy-Chapman-Stern 水相扩散双层理论能够非常好地描述庆大霉素和精胺与在0.1 M NaCl溶液中形成的双层膜的相互作用。因此，该理论对于描述这些阳离子与生物膜双层成分的相互作用应该是有用的，生物膜通常含有少于50%的带负电脂质。例如，我们的结果支持了 Sastrasinh 等人 [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350 - 358] 的观点，即磷脂酰肌醇是肾刷状缘膜中庆大霉素的主要结合位点。
