Enzymatic methylation of arsenic compounds: assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver.
Enzymatic methylation of arsenic compounds: assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver.
复制标题
砷化合物的酶促甲基化：兔肝亚砷酸甲基转移酶和单甲基胂酸甲基转移酶的测定、部分纯化和性质。
DOI：
10.1021/tx00050a006
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发表时间：
1995
影响因子：
4.1
通讯作者：
Aposhian,HV
中科院分区：
文献类型：
--
作者：
Zakharyan,R;Wu,Y;Bogdan,GM;Aposhian,HV
已经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，用于兔肝甲基转移酶对亚砷酸盐和单甲基胂酸盐 (MMA) 的甲基化。通过使用氯仿萃取、离子交换色谱、TLC 或 HPLC 分离、鉴定和测量反应产物，对测定进行了验证。参与该途径的酶，亚砷酸甲基转移酶和 MMA 甲基转移酶，已从兔肝脏中纯化了约 2000 倍。凝胶电泳后，获得一条带有两种酶活性的条带。纯化的亚砷酸甲基转移酶和单甲基胂酸甲基转移酶的最适pH分别为8.2和8.0。部分纯化的亚砷酸甲基转移酶催化合成胂酸单甲酯，需要硫醇、S-腺苷甲硫氨酸和亚砷酸。催化单甲基胂酸甲基化为二甲基胂酸的不同酶活性也需要SAM和硫醇。尽管亚砷酸甲基转移酶和单甲基胂酸甲基转移酶具有不同的底物、最适pH和底物饱和浓度，但这两种活性是否存在于一个蛋白质分子或不同的蛋白质分子上仍然不确定。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。砷酸盐、硒酸盐、亚硒酸盐或硒化物均不会被纯化的酶制剂甲基化。使用粗提物（通常称为胞质溶胶）来研究这些处理砷物质的甲基转移酶的特性时应谨慎对待，因为此类粗提物含有抑制活性和其他干扰活性。
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. Theassay 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 monomethylar-sonate. A different enzyme activity that catalyzes themethylation of monomethylarsonateto 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 calledcytosol, 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.