Kalinoski D L, Bryant B P, Shaulsky G, Brand J G, Harpaz S
Monell Chemical Senses Center, Philadelphia, PA 19104.
Brain Res. 1989 May 29;488(1-2):163-73. doi: 10.1016/0006-8993(89)90705-1.
We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.
我们在此报告精氨酸结合位点的特性以及相关的神经生理学研究。通过对克鲁格和卡根方法的改进，测量了L-[³H]精氨酸与味觉上皮细胞P2组分的结合。确定了测量最大结合活性的参数，包括孵育时间和培养基pH值。在pH 7.8时，4℃下L-[³H]精氨酸与P2组分结合的表观单缔合速率常数（kobs）为4.72×10⁵ M⁻¹·min⁻¹。解离过程较为复杂，产生两个解离速率常数，分别为1.77 min⁻¹和8.34×10⁻³ min⁻¹。这些数据表明L-精氨酸存在两种亲和力状态。根据k⁻¹/k⁺¹计算得到的KD值分别为1.3×10⁻⁶ M和1.8×10⁻⁸ M。L-精氨酸结合的同源抑制研究不适合简单的质量作用关系（希尔系数为0.79），但最适合双位点模型，高亲和力状态的IC50值为1.6×10⁻⁶ M，低亲和力状态的IC50值为9×10⁻⁴ M。多单位神经记录研究了多种含胍化合物的刺激效果。与L-精氨酸相比，只有L-精氨酸甲酯和L-α-氨基-β-胍基丙酸（L-AGPA）是有效的刺激物。交叉适应实验表明，在10⁻⁴ M的L-精氨酸甲酯、L-AGPA以及程度稍低的D-精氨酸作用下，对10⁻⁶ M的L-精氨酸具有有效的交叉适应刺激作用。在竞争结合研究中，L-精氨酸甲酯、L-AGPA和D-精氨酸也能抑制10⁻⁶ M的L-[³H]精氨酸的结合，但每种物质只识别一种亲和力状态。交叉适应能力较差的刺激物L-谷氨酸、甘氨酸和L-丙氨酸仅在浓度高于10⁻³ M时才会产生抑制作用，这表明L-精氨酸的结合位点具有选择性。这些研究表明，L-精氨酸结合至少存在两种亲和力状态，结合位点具有特异性，且L-精氨酸受体的有效激动剂必须含有胍基和未被阻断的L-α-氨基。
