Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems:: Comparison with the recombinant enzyme purified from Escherichia coli
Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems:: Comparison with the recombinant enzyme purified from Escherichia coli
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DOI：
10.1006/prep.1999.1030
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发表时间：
1999-06-01
影响因子：
1.6
通讯作者：
Coughtrie, MWH
中科院分区：
文献类型：
--
作者：
Dajani, R;Sharp, S;Coughtrie, MWH
硫酸化是一种主要的代谢途径，由硫转移酶家族成员催化，调节许多内源性和外源性化学物质的生物活性。许多这些酶已经在原核和真核系统中表达，以产生用于生化和物理表征的蛋白质。然而，有效地利用异种表达系统来产生用于这种目的的重组酶取决于所表达的蛋白质忠实地代表“天然”蛋白质。对于人硫转移酶，尽管重组酶被广泛使用，但很少有人关注这一点。通过比较细胞提取物中重组酶与人血小板中存在的酶的K-m值，以及大肠杆菌表达后纯化的重组酶的K-m值，我们验证了多种用于生产人多巴胺代谢硫转移酶SULT1A3的异源表达系统，包括大肠杆菌、酿酒酵母、COS-7和V79细胞。这是首次报道细胞质硫转移酶在酵母中的异源表达。SULT1A3在所有细胞类型中均有表达，在所研究的所有异种系统中，多巴胺的K-m在所应用的条件下约为1 μ M，与人血小板测定的值相比较有利。我们还通过SDS-PAGE、电喷雾电离质谱、动态光散射和沉淀分析确定了纯化的重组酶的亚基和天然分子量。经光散射和沉淀分析，在大肠杆菌中表达后纯化的酶以同型二聚体存在，其M-r约为68,000。质谱分析显示两种物质的实验测定质量分别为34,272和34,348，它们对应于具有一个或两个2-巯基乙醇加合物的天然蛋白质。我们得出结论，在原核和真核异种系统中表达的酶，以及从大肠杆菌中纯化的酶，等同于在人体组织制剂中发现的酶，(C) 1999学术出版社。
Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of K-m values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the K-m for dopamine under the conditions applied was approximately 1 mu M in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with M-r approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations, (C) 1999 Academic Press.