Miao H H, Ye J S, Wong S L, Wang B X, Li X Y, Sheu F S
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, People's Republic of China.
Bioelectrochemistry. 2000 Jun;51(2):163-73. doi: 10.1016/s0302-4598(00)00062-3.
Neurogranin (Ng) is a neuron-specific protein kinase C (PKC) substrate, which contains four cysteine (Cys) residues. Recently, it has been shown that Ng is a redox-sensitive protein and is a likely target of nitric oxide (NO) and other oxidants [F.-S. Sheu, C.W. Mahoney, K. Seki, K.-P. Huang, Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin, J. Biol. Chem. 271 (1996) 22407-22413: J. Li, J.H. Pak, F.L. Huang, K.-P. Huang, N-methyl-D-aspartate induces neurogranin/RC3 oxidation in rat brain slices, J. Biol. Chem. 274 (1999) 1294-1300]. In this study, we directly examine the redox reactions between dissolved NO and Cys as well as between NO and bacterial expressed Ng in its reduced form, at concentrations approximate to the physiological levels in phosphate buffer solution (PBS) under aerobic conditions. The reaction kinetics are measured directly by our newly developed electrochemical sensor. Our sensor is based on the chemical modification of electrode with immobilized nanoparticles of transition metal palladium (Pd) which serves as catalytic centers for the electrochemical oxidation of thiol and NO selectively and quantitatively at different potentials. It detects Cys and Ng in a linear range from nano to micromolar concentration at + 450 mV, vs. a saturated calomel reference electrode (SCE), while the detection of NO at the sensor can be optimally achieved at + 700 mV (vs. SCE) with a linear current-to-concentration range of nM to microM. It thus provides a selective control to monitor two reactants independently. With this sensor as a detector, we found that (1) the oxidation of either Cys or Ng by NO is a fast reaction which reaches a near completion within 1-2 min at its physiological concentration; and (2) after the completion of reaction, NO is mostly, if not all, regenerated, an observation consistent with the reaction mechanism involving the formation of S-nitrosothiol as an intermediate. The reaction kinetics of both NO to Cys and NO to Ng implies that NO can achieve local action on cellular proteins in addition to its effect on targets located in neighboring cells via concentration-gradient-dependent diffusion.
神经颗粒素（Ng）是一种神经元特异性蛋白激酶C（PKC）底物，含有四个半胱氨酸（Cys）残基。最近的研究表明，Ng是一种对氧化还原敏感的蛋白质，可能是一氧化氮（NO）和其他氧化剂的作用靶点[F.-S. 谢，C.W. 马奥尼，K. 关，K.-P. 黄，大鼠脑内神经颗粒素的一氧化氮修饰影响其蛋白激酶C磷酸化及对钙调蛋白的亲和力，《生物化学杂志》271卷（1996年）第22407 - 22413页；J. 李，J.H. 朴，F.L. 黄，K.-P. 黄，N - 甲基 - D - 天冬氨酸诱导大鼠脑片神经颗粒素/RC3氧化，《生物化学杂志》274卷（1999年）第1294 - 1300页]。在本研究中，我们直接检测了在有氧条件下，溶解态NO与Cys之间以及NO与还原形式的细菌表达Ng之间在磷酸盐缓冲溶液（PBS）中接近生理水平浓度时的氧化还原反应。反应动力学通过我们新开发的电化学传感器直接测量。我们的传感器基于用固定化过渡金属钯（Pd）纳米颗粒对电极进行化学修饰，Pd纳米颗粒作为催化中心，可在不同电位下选择性且定量地对硫醇和NO进行电化学氧化。它在相对于饱和甘汞参比电极（SCE）为 + 450 mV时，能检测纳摩尔到微摩尔浓度范围内呈线性变化的Cys和Ng，而在 + 700 mV（相对于SCE）时可最佳地检测传感器中的NO，线性电流 - 浓度范围为纳摩尔到微摩尔。因此，它提供了一种选择性控制，能够独立监测两种反应物。以该传感器作为检测器，我们发现：（1）在生理浓度下，NO对Cys或Ng的氧化是一个快速反应，在1 - 2分钟内接近完成；（2）反应完成后，NO即使不是全部也大多会再生，这一观察结果与涉及形成亚硝基硫醇作为中间体的反应机制一致。NO与Cys以及NO与Ng的反应动力学表明，NO除了通过浓度梯度依赖扩散作用于邻近细胞中的靶点外，还能对细胞内蛋白质产生局部作用。
