Erickson J, Goldstein B, Holowka D, Baird B
Department of Chemistry, Cornell University, Ithaca, New York 14853.
Biophys J. 1987 Oct;52(4):657-62. doi: 10.1016/S0006-3495(87)83258-7.
For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.
对于与细胞表面受体相互作用的单价配体，我们直接观察到了正向速率常数对每个细胞受体数量（N）的功能依赖性。我们研究的实验系统由单价配体2,4 - 二硝基苯基（DNP）-氨基己酰 - L - 酪氨酸（DCT）与结合在大鼠嗜碱性白血病（RBL）细胞高亲和力受体上的二价单克隆抗DNP免疫球蛋白E（IgE）组成。为了测量DNP对抗体结合位点的占据分数，我们采用了一种最近开发的荧光技术（埃里克森，J.，凯恩，B.，戈尔茨坦，D.，霍洛卡，D.，和贝尔德，B.，1986，《分子免疫学》，72:769 - 781）。我们的结果很好地符合方程（伯格和珀塞尔，1977，《生物物理学杂志》，20:193 - 219）konc = 4πDaNκon / [4πDa + Nκon]，其中konc是与细胞结合的正向速率常数，D是配体的扩散常数，a是细胞半径，κon是描述单个IgE结合位点 - DNP相互作用的内在正向速率常数。如果D固定为10^(-5) cm²/s，累积数据的最佳拟合预测平均细胞半径约为4微米，κon约为1.8×10^(-13) cm³/s [1.1×10^(8)(M·s)^(-1)]；这两个值分别与RBL细胞大小以及DCT与溶液中IgE结合的单位点正向速率常数非常吻合。我们相信这是第一份直接说明表面密度在决定小分子与膜受体结合速率方面作用的实验证据报告。
