Spinedi A, Pacini L, Luly P
Department of Biology, University of Rome Tor Vergata, Italy.
Biochem J. 1989 Jul 15;261(2):569-73. doi: 10.1042/bj2610569.
The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.
研究了局部麻醉药普鲁卡因、丁卡因和利多卡因以及抗抑郁药丙咪嗪对人红细胞乙酰胆碱酯酶的影响。所有这四种两亲性药物均抑制酶活性，普鲁卡因的IC50（引起50%抑制的浓度）约为0.40 mM，丁卡因的IC50为0.05 mM，丙咪嗪的IC50为0.70 mM，利多卡因的IC50为7.0 mM。通过稳态荧光偏振评估，普鲁卡因和丁卡因在不干扰膜脂质环境物理状态的浓度下竞争性抑制乙酰胆碱酯酶活性，而利多卡因和丙咪嗪在诱导膜流动性增强的浓度下表现出混合抑制动力学。研究了膜完整性是否是丙咪嗪和利多卡因发挥作用的先决条件这一问题。用1% Triton X-100使膜增溶，并通过稀释将去污剂浓度降至0.05%[高于Triton X-100临界胶束浓度（c.m.c.）]，这并未显著影响这两种两亲性药物在其IC50时的抑制效力；在去污剂浓度增加的情况下，丙咪嗪的抑制效力逐渐降低，但未消除，而利多卡因的抑制作用仅略有减弱，即使在1% Triton X-100时也是如此。研究表明，竞争性和混合抑制动力学均不是由脂质环境物理状态的改变驱动的蛋白质构象变化引起的，而是由两亲性药物与乙酰胆碱酯酶的直接相互作用引起的。特别是，在Triton X-100浓度远高于其c.m.c.时观察到的丙咪嗪和利多卡因抑制效力的部分丧失，可以用两亲分子在去污剂胶束中的分配来解释，进而可以用两种抑制剂在水相中的有效浓度降低来解释。
