Zakharyan R, Wu Y, Bogdan G M, Aposhian H V
Department of Molecular & Cellular Biology, University of Arizona, Tucson 85721, USA.
Chem Res Toxicol. 1995 Dec;8(8):1029-38. doi: 10.1021/tx00050a006.
A rapid, accurate, in vitro assay utilizing radioactive S-adenosylmethionine (SAM) has been developed for the methylation of arsenite and monomethylarsonate (MMA) by rabbit liver methyltransferases. The assay has been validated by separating, identifying, and measuring the products of the reaction using chloroform extraction, ion exchange chromatography, TLC, or HPLC. The enzymes involved in this pathway, arsenite methyltransferase and MMA methyltransferase, have been purified approximately 2000-fold from rabbit liver. After gel electrophoresis, a single band is obtained with both enzyme activities in it. The pH optima for purified arsenite methyltransferase and monomethylarsonic acid methyltransferase are 8.2 and 8.0, respectively. A thiol, S-adenosylmethionine, and arsenite are required for the partially purified arsenite methyltransferase that catalyzes the synthesis of monomethylarsonate. A different enzyme activity that catalyzes the methylation of monomethylarsonate to dimethylarsinate also requires SAM and a thiol. Even though arsenite methyltransferase and monomethylarsonate methyltransferase have different substrates, pH optima, and saturation concentrations for their substrates, whether the two activities are present on one protein molecule or different protein molecules is still uncertain. Both activities have a molecular mass of 60 kDa as determined by gel exclusion chromatography. There is no evidence at the present time for these enzyme activities being on different protein molecules. Neither arsenate, selenate, selenite, or selenide are methylated by the purified enzyme preparations. Results from the use of crude extracts, often called cytosol, to study the properties of these methyltransferases dealing with arsenic species should be viewed with caution since such crude extracts contain inhibiting and other interfering activities.
已开发出一种利用放射性S-腺苷甲硫氨酸（SAM）的快速、准确的体外测定方法，用于检测兔肝甲基转移酶对亚砷酸盐和一甲基胂酸（MMA）的甲基化作用。通过使用氯仿萃取、离子交换色谱、薄层层析（TLC）或高效液相色谱（HPLC）分离、鉴定和测量反应产物，对该测定方法进行了验证。参与此途径的酶，即亚砷酸盐甲基转移酶和MMA甲基转移酶，已从兔肝中纯化了约2000倍。凝胶电泳后，得到一条含有两种酶活性的单一谱带。纯化后的亚砷酸盐甲基转移酶和一甲基胂酸甲基转移酶的最适pH分别为8.2和8.0。催化合成一甲基胂酸的部分纯化的亚砷酸盐甲基转移酶需要一种硫醇、S-腺苷甲硫氨酸和亚砷酸盐。另一种催化一甲基胂酸甲基化生成二甲基胂酸的酶活性也需要SAM和一种硫醇。尽管亚砷酸盐甲基转移酶和一甲基胂酸甲基转移酶的底物、最适pH以及底物的饱和浓度不同，但这两种活性是存在于一个蛋白质分子上还是不同的蛋白质分子上仍不确定。通过凝胶排阻色谱法测定，两种活性的分子量均为60 kDa。目前没有证据表明这些酶活性存在于不同的蛋白质分子上。纯化的酶制剂均未使砷酸盐、硒酸盐、亚硒酸盐或硒化物发生甲基化。由于此类粗提物含有抑制性和其他干扰活性，因此在使用粗提物（通常称为胞质溶胶）研究这些处理砷物种的甲基转移酶的特性时，所得结果应谨慎看待。
