Gabev E, Kasianowicz J, Abbott T, McLaughlin S
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
Biochim Biophys Acta. 1989 Feb 13;979(1):105-12. doi: 10.1016/0005-2736(89)90529-4.
Schacht (Schacht, J. (1976) J. Neurochem. 27, 1119-1124) demonstrated that neomycin, an aminoglycoside antibiotic, binds with high affinity to phosphatidylinositol 4,5-bisphosphate (PIP2). We investigated the binding of neomycin to PIP2 by making electrophoretic mobility measurements with multilamellar bilayer vesicles and surface potential measurements with monolayers. The bilayers and monolayers were formed from mixtures of PIP2 and egg phosphatidylcholine (PC) in 0.1 M KCl at pH 7. Neomycin does not bind to PC; 10(-3) M neomycin affects neither the zeta potential of PC vesicles nor the surface potential of PC monolayers. In contrast, 10(-6) M neomycin reduces the magnitude of the zeta potential of PC/PIP2 vesicles (5, 9, and 17 mol% PIP2) and the surface potential of monolayers (17 mol% PIP2) to less than 50% of their initial values. The electrophoretic mobility results indicate that neomycin forms an electroneutral complex with PIP2; high concentrations (greater than 10(-4) M) of neomycin reduce the zeta potential of the PC/PIP2 vesicles to zero. We could describe our data with the Gouy-Chapman-Stern theory assuming the intrinsic association constant of the 1:1 neomycin-PIP2 complex is 10(5) M-1. Neomycin is widely used in cell biology to interfere with the generation of second messengers; we discuss the relevance of our results to these studies. Specifically, 10(-6) M neomycin binds greater than 50% of the PIP2 in a bilayer or monolayer but 10(-5)-10(-3) M neomycin is required to affect the turnover of PIP2 in permeabilized platelets, mast cells, and sea urchin eggs. This result is consistent with a hypothesis that most of the PIP2 in the inner leaflet of these plasma membranes is not accessible to neomycin because it is associated with proteins.
沙赫特（沙赫特，J.（1976年）《神经化学杂志》27卷，1119 - 1124页）证明，氨基糖苷类抗生素新霉素能与磷脂酰肌醇4,5 - 二磷酸（PIP2）高亲和力结合。我们通过用多层双层囊泡进行电泳迁移率测量以及用单层膜进行表面电位测量来研究新霉素与PIP2的结合。双层膜和单层膜由PIP2与鸡蛋磷脂酰胆碱（PC）在pH值为7的0.1 M氯化钾中的混合物形成。新霉素不与PC结合；10⁻³ M新霉素既不影响PC囊泡的ζ电位，也不影响PC单层膜的表面电位。相比之下，10⁻⁶ M新霉素会使PC/PIP2囊泡（含5%、9%和17% PIP2）的ζ电位幅度以及单层膜（含17% PIP2）的表面电位降低至初始值的50%以下。电泳迁移率结果表明新霉素与PIP2形成了电中性复合物；高浓度（大于10⁻⁴ M）的新霉素会使PC/PIP2囊泡的ζ电位降至零。假设新霉素 - PIP2 1:1复合物的固有缔合常数为10⁵ M⁻¹，我们可以用古伊 - 查普曼 - 斯特恩理论来描述我们的数据。新霉素在细胞生物学中被广泛用于干扰第二信使的产生；我们讨论了我们的结果与这些研究的相关性。具体而言，10⁻⁶ M新霉素能结合双层膜或单层膜中超过50%的PIP2，但在通透的血小板、肥大细胞和海胆卵中，需要10⁻⁵ - 10⁻³ M新霉素才能影响PIP2的周转。这一结果与以下假设一致：这些质膜内小叶中的大多数PIP2因与蛋白质结合而无法被新霉素接触到。
