Sutter A, Riopelle R J, Harris-Warrick R M, Shooter E M
J Biol Chem. 1979 Jul 10;254(13):5972-82.
Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.
对来自8日龄鸡胚感觉神经节的单细胞进行的125I-β神经生长因子（NGF）结合的稳态和动力学研究表明，存在两个不同的、可饱和的结合位点，其解离常数分别为Kd(I)=2.3×10(-11)M和Kd(II)=1.7×10(-9)M。亲和力的差异是由于不同的解离速率常数（k-1(I)=10(-3)s-1，k-1(II)=2×10(-1)s-1）。与两个位点的结合显然是扩散控制的（k+1(I)=4.8×10(7)M-1s-1，k+2(II)=10(7)至10(8)M-1s-1）。βNGF与两个位点的结合是特异性的，因为所测试的多种激素或蛋白质均不竞争125I-βNGF与这两个位点中任何一个的结合。125I-βNGF结合的异质性不是由于125I-βNGF制剂的异质性，也不是由于负协同结合。在通过添加饱和量的未标记βNGF诱导125I-βNGF解离的实验中，以两种解离速率常数之一释放的125I-βNGF的比例仅取决于解离开始前两个位点的占有率，而与解离过程中位点的总占有率无关。在占有率增加和降低的条件下，未标记的βNGF都会加速125I-βNGF从高亲和力结合位点I的解离速率。尽管125I-βNGF的解离特性会随着细胞与配体接触时间的增加而变化，并且125I-βNGF在与细胞结合后会降解，但这些次级过程不会干扰结合数据的分析。在用于分析的125I-βNGF的最低浓度下，不到10%的125I-βNGF会降解。动力学和稳态结合数据在2℃以及37℃时均揭示了两个NGF结合位点。
