Gu Y, Lee H, Kirchhoff J R, Manzey L, Hudson R A
Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43606.
Biochemistry. 1994 Jul 19;33(28):8486-94. doi: 10.1021/bi00194a013.
The synthesis of 4- and 5-hydroxy-3-[(trimethylammonio)methyl]catechol (4- and 5-HTMC) was carried out to examine their proposed involvement as intermediates in the spontaneous redox-dependent half-of-sites inactivation of neurotoxin binding sites in the nicotinic acetylcholine receptor (nAcChR) mediated by the parent compound 3-[(trimethylammonio)methyl]catechol (TMC) [Nickoloff et al. (1985) Biochemistry 24, 999-1007]. Oxidation of 4- and 5-HTMC occurred with sodium periodate with facile conversion to the corresponding p-quinones which were intercepted with thiols and cyclopentadiene. Both 4- and 5-HTMC inactivated neurotoxin binding in the nAcChR in a time course and over a concentration range consistent with their involvement as intermediates in the TMC redox-dependent inactivation of neurotoxin ([125I]-alpha-bungarotoxin) binding sites. Rapid concentration-dependent inactivation of neurotoxin sites occurred over a 10-1000 microM range and was resistant to further inactivation after 50% loss of available toxin binding sites on the nAcChR. Both 4- and 5-HTMC inactivated nAcChR neurotoxin sites much more rapidly and efficiently than was observed previously with TMC. The apparent binding constants for 4- and 5-HTMC with the nAcChR, calculated from their concentration-dependent inactivation behavior toward toxin binding sites, were Kd = 224 +/- 98 and 39 +/- 17 microM, respectively. The observed results and the redox potentials (vs Ag/AgCl reference electrode) measured by cyclic voltammetry at pH 1.8 for TMC (719 mV) and the 4- and 5-HTMC derivatives (519 and 443 mV, respectively) supported the previously proposed mechanism for inactivation of the nAcChR by TMC.(ABSTRACT TRUNCATED AT 250 WORDS)
合成了4-和5-羟基-3-[(三甲基铵基)甲基]儿茶酚（4-和5-HTMC），以检验它们作为中间体参与由母体化合物3-[(三甲基铵基)甲基]儿茶酚（TMC）介导的烟碱型乙酰胆碱受体（nAcChR）中神经毒素结合位点的自发氧化还原依赖性半位点失活的可能性[尼科洛夫等人（1985年），《生物化学》24卷，999 - 1007页]。4-和5-HTMC用高碘酸钠氧化，能轻易转化为相应的对醌，这些对醌可被硫醇和环戊二烯截获。4-和5-HTMC在时间进程和浓度范围内均使nAcChR中的神经毒素结合失活，这与它们作为TMC氧化还原依赖性神经毒素（[125I]-α-银环蛇毒素）结合位点失活的中间体的情况相符。在10 - 1000微摩尔范围内，神经毒素位点迅速发生浓度依赖性失活，且在nAcChR上可用毒素结合位点损失50%后对进一步失活具有抗性。4-和5-HTMC使nAcChR神经毒素位点失活的速度和效率比之前观察到的TMC快得多。根据它们对毒素结合位点的浓度依赖性失活行为计算得出，4-和5-HTMC与nAcChR的表观结合常数分别为Kd = 224±98微摩尔和39±17微摩尔。观察结果以及在pH 1.8下通过循环伏安法测得的TMC（719毫伏）以及4-和5-HTMC衍生物（分别为519和443毫伏）的氧化还原电位（相对于Ag/AgCl参比电极）支持了先前提出的TMC使nAcChR失活的机制。（摘要截断于250字）
