采用电子光谱、荧光光谱、循环伏安法研究了[Cu(phendio)(L-Phe)(H_2O)](ClO_4)·H_2O(简称CuPP,phendio=1,10-菲咯啉-5,6-二酮,L-Phe=L-苯丙氨酸)和小牛胸腺 DNA 之间的相互作用.结果表明,当加入一定量的 DNA 时,电子光谱的最大吸收峰明显红移,产生减色效应;循环伏安法中配合物的氧化还原峰电流明显降低.同时,配合物也能较大程度地淬灭 EB-DNA 复合物的荧光,证明配合物与 DNA 存在插入结合.凝胶电泳法研究发现,该配合物在抗坏血酸和 H_2O_2的存在下能有效地将超螺旋型质粒 pBR322 DNA 切割为缺刻型 DNA 和线型 DNA.
Several vanadium compounds have been known for the hypoglycemic and anticancer effects. However, the mechanisms of the pharmacological and toxicological effects were not clear. In this work, we in- vestigated the potential targets of vanadium in mitochondria. Vanadyl ions were found to bind to mi- tochondria from rat liver with a stoichiometry of 244±58 nmol/mg protein and an apparent dissocia- tion constant (Kd) of (2.0±0.8)×10·16 mol/L. Using size exclusion chromatography, a vanadium-binding protein was isolated and identified to be the 60-kDa heat shock protein (HSP60) by mass spectrometry analysis and immunoassays. Additionally, binding of vanadyl ions was found to result in depolymeri- zation of homo-oligomeric HSP60 (GroEL). HSP60 is an indispensable molecular chaperone and in- volved in many kinds of pathogenesis of inflammatory and autoimmune diseases, e.g. type 1 diabetes. Our results suggested that HSP60 could be a novel important target involved in the biological and/or toxicological effects of vanadium compounds.
LEI WanHuaLIU HuiXueZHONG LiJunYANG XiaoDaWANG Kui
p21Waf1/Cip1, best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p21 are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical properties have been comparatively sparse. A human p21 gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109. Recombinant protein was expressed as a non-fusion protein and purified by gel filtration and anion exchange chromatography. The purified protein was verified by Western blot and the functional activity was recognized by pull-down assay. Furthermore, circular dichroism, fluorescence spectroscopy, and fluorescence quenching methods were used to characterize the conformational properties of the purified protein. The results indicate that it was largely unstructured under the native solution conditions, and its tryptophan residues were exposed and located in a positively charged microenvironment. This study lays a good foundation for further study of p21 binding to its different partners.
SHI Qiao-yunZHENG Yong-chenREN Jin-songQU Xiao-gang
