Thomas T P, Wang E, Pfeiffer D R, Taylor R W
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Arch Biochem Biophys. 1997 Jun 15;342(2):351-61. doi: 10.1006/abbi.1997.0121.
Ionophore A23187 has been proposed to form Ca(2+)- conducting channels that arise from dimers and oligomers of the compound (e.g., Balasubramanian, S. V., and Easwaran, K. R. K. (1989) Biochem. Biophys. Res. Commun. 158, 891-897). To investigate this possibility, the solution behavior of A23187 in chloroform, n-hexane, ethanol, 80% methanol-water, and palmitoyloleoylphosphatidyl choline (POPC) vesicles was investigated using UV-VIS, circular dichroism (CD), and 1H NMR techniques. The concentration dependence of the UV-VIS and CD spectra obtained in freshly prepared chloroform solutions indicates that neutral A23187 (HA) exists as a monomer for ionophore concentrations in the range of 50-1000 microM. The cause of time- and concentration-dependent spectral alterations which gave rise to the dimer/channel hypothesis was also investigated. For solutions of 50-1000 microM A23187 in chloroform, n-hexane, and ethanol stored in the dark, no spectral changes were observed for periods of 2 months. However, solutions in these solvents did show time-dependent spectral changes when exposed to light. In 80% methanol-water or phospholipid vesicles, similar spectral changes were observed, even when the solutions were protected from light. Application of TLC and MS methods indicate that the time-dependent spectral changes reflect degradation of A23187, not dimer or oligomer formation. The degradative processes proceed with half-lives ranging from approximately 75 to > 400 h, and are influenced by several factors, including solvent, exposure to light, ionophore concentration, pH, and the presence of metal ions, EDTA, dissolved oxygen, and a radical inhibitor. The kinetics of Ca2+ transport into Quin-2-loaded POPC vesicles by authentic A23187 give no evidence of a channel mechanism, even following a previous and lengthy coincubation of the ionophore with the vesicles.
离子载体A23187被认为可形成由该化合物的二聚体和寡聚体产生的Ca(2+)传导通道（例如，Balasubramanian, S. V., 和Easwaran, K. R. K. (1989) Biochem. Biophys. Res. Commun. 158, 891 - 897）。为了研究这种可能性，使用紫外可见光谱（UV - VIS）、圆二色光谱（CD）和1H核磁共振（NMR）技术研究了A23187在氯仿、正己烷、乙醇、80%甲醇 - 水以及棕榈酰油酰磷脂酰胆碱（POPC）囊泡中的溶液行为。在新制备的氯仿溶液中获得的UV - VIS和CD光谱的浓度依赖性表明，对于浓度在50 - 1000 microM范围内的离子载体，中性A23187（HA）以单体形式存在。还研究了导致二聚体/通道假说的时间和浓度依赖性光谱变化的原因。对于在黑暗中储存的50 - 1000 microM A23187在氯仿、正己烷和乙醇中的溶液在2个月内未观察到光谱变化。然而，当暴露于光时，这些溶剂中的溶液确实显示出时间依赖性光谱变化。在80%甲醇 - 水或磷脂囊泡中，即使溶液避光，也观察到类似的光谱变化。薄层层析（TLC）和质谱（MS）方法的应用表明，时间依赖性光谱变化反映的是A23187的降解，而非二聚体或寡聚体的形成。降解过程的半衰期约为75至>400小时，并受多种因素影响，包括溶剂、光照、离子载体浓度、pH值以及金属离子、乙二胺四乙酸（EDTA）、溶解氧和自由基抑制剂的存在。即使在离子载体与囊泡预先长时间共孵育之后，真正的A23187将Ca2+转运到负载喹啉 - 2的POPC囊泡中的动力学也没有提供通道机制的证据。
