Styblo M, Thomas D J
Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270, USA.
Toxicol Appl Pharmacol. 1997 Nov;147(1):1-8. doi: 10.1006/taap.1997.8256.
The dynamics of interactions between rat liver cytosolic proteins and arsenicals were examined in an in vitro methylation system that contained cytosol, glutathione, S-adenosylmethionine, and 1 microM -73As-arsenite. After incubation at 37 degrees C for up to 90 min, low-molecular-weight components of the assay system (<10 kDa) were removed by ultrafiltration and cytosolic proteins were separated by size-exclusion chromatography on Sephacryl S-300 gel. Five 73As-labeled protein peaks were found in chromatographic profiles. The estimated molecular masses of 73As-labeled proteins eluting in the three earliest peaks were as follows: Vo, >/=1000 kDa; A, 135 kDa; and B, 38 kDa. Peak C eluted immediately before the total volume (VT) of the chromatographic column; peak D eluted after the VT. 73As bound to proteins was released by CuCl treatment and speciated by thin-layer chromatography. Amounts and ratios of inorganic As, methyl As, and dimethyl As associated with cytosolic proteins depended upon the incubation interval. Inorganic As was present in all protein peaks. Methyl As was primarily associated with peaks A and C; dimethyl As was associated with peaks B and C. To examine the effect of valence on the binding of methylarsenicals to cytosolic proteins, trivalent or pentavalent 14C-labeled methyl As or dimethyl As was incubated in an in vitro system designed to minimize the enzymatically catalyzed production of methylated arsenicals. Proteins in peaks A, B, and C bound preferentially trivalent methyl and dimethyl As. Peak D bound either trivalent or pentavalent methyl and dimethyl As. Protein-bound inorganic and methyl As were substrates for the production of dimethyl As in an in vitro methylation system, suggesting a role for protein-bound arsenicals in the biomethylation of this metalloid.
在一个包含胞质溶胶、谷胱甘肽、S-腺苷甲硫氨酸和1微摩尔-73As-亚砷酸盐的体外甲基化系统中，研究了大鼠肝脏胞质溶胶蛋白与砷化合物之间的相互作用动力学。在37℃孵育长达90分钟后，通过超滤去除测定系统的低分子量组分（<10 kDa），并通过在Sephacryl S-300凝胶上的尺寸排阻色谱法分离胞质溶胶蛋白。在色谱图中发现了五个73As标记的蛋白峰。在最早的三个峰中洗脱的73As标记蛋白的估计分子量如下：Vo，≥1000 kDa；A，135 kDa；B，38 kDa。峰C在色谱柱总体积（VT）之前立即洗脱；峰D在VT之后洗脱。与蛋白结合的73As通过CuCl处理释放，并通过薄层色谱法进行形态分析。与胞质溶胶蛋白相关的无机砷、甲基砷和二甲基砷的量和比例取决于孵育时间间隔。无机砷存在于所有蛋白峰中。甲基砷主要与峰A和C相关；二甲基砷与峰B和C相关。为了研究价态对甲基砷化合物与胞质溶胶蛋白结合的影响，将三价或五价的14C标记甲基砷或二甲基砷在一个旨在尽量减少酶催化产生甲基化砷化合物的体外系统中孵育。峰A、B和C中的蛋白优先结合三价甲基砷和二甲基砷。峰D结合三价或五价甲基砷和二甲基砷。蛋白结合的无机砷和甲基砷是体外甲基化系统中产生二甲基砷的底物，表明蛋白结合的砷化合物在这种类金属的生物甲基化中起作用。
