Si Yue-Xiu, Lee Jinhyuk, Zhao Feng, Yin Shang-Jun, Park Yong-Doo, Qian Guo-Ying, Jiang Xia-Min
a School of Marine Sciences , Ningbo University , Ningbo 315211 , P.R. China.
b College of Biological and Environmental Sciences, Zhejiang Wanli University , Ningbo 315100 , P.R. China.
J Biomol Struct Dyn. 2016 Aug;34(8):1763-77. doi: 10.1080/07391102.2015.1091747. Epub 2015 Oct 19.
Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.
精氨酸激酶与海洋无脊椎动物适应高盐度和重金属离子水平等环境胁迫密切相关。在本研究中，研究了镉离子（Cd(2+)）对法老乌贼（Sepia pharaonis）精氨酸激酶（SPAK）的影响。从法老乌贼的肌肉中分离出SPAK，进一步纯化后，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上显示为单一条带。Cd(2+)有效地使SPAK失活，双倒数动力学表明Cd(2+)对精氨酸和ATP诱导非竞争性抑制。荧光光谱法结果表明，Cd(2+)诱导SPAK的三级结构发生变化，疏水表面暴露，直接诱导SPAK聚集。添加渗透剂、甘氨酸和脯氨酸成功地阻止了SPAK聚集，并恢复了SPAK的构象和活性。涉及SPAK和Cd(2+)的分子动力学模拟表明，Cd(2+)部分阻断了ATP进入活性位点，这一结果与显示Cd(2+)诱导SPAK失活的实验结果一致。这些结果证明了Cd(2+)对SPAK酶功能和去折叠的影响，包括聚集以及渗透剂对SPAK折叠的保护作用。本研究提供了Cd(2+)在代谢酶SPAK背景下毒性的具体证据，并阐明了重金属在乌贼中的毒性作用和解毒机制。
