Tetrahydrobiopterin-dependent production of l-DOPA in NRK fibroblasts transfected with tyrosine hydroxylase cDNA: future use for intracerebral grafting
Tetrahydrobiopterin-dependent production of l-DOPA in NRK fibroblasts transfected with tyrosine hydroxylase cDNA: future use for intracerebral grafting
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酪氨酸羟化酶 cDNA 转染的 NRK 成纤维细胞中四氢生物蝶呤依赖性左旋多巴的产生：未来用于脑内移植
DOI：
10.1016/0304-3940(90)90008-w
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发表时间：
1990
影响因子：
2.5
通讯作者：
S. Kohsaka
中科院分区：
文献类型：
--
作者：
K. Uchida;A. Ishii;N. Kaneda;S. Toya;T. Nagatsu;S. Kohsaka
在本研究中，酪氨酸羟化酶(TH; EC 1.14。16.2)将cDNA转染培养成纤维细胞，测定l- 3,4 -二羟基苯丙氨酸（l-DOPA）的产量。将2型TH cDNA转染到正常大鼠肾脏成纤维细胞（NRK-49F）中，通过免疫细胞化学染色和TH抗血清免疫印迹分析筛选TH基因的表达。获得了几个产生TH分子的NRK转基因克隆。在一个完整的体外分析系统中，表达的TH分子显示出较高的酶活性。然而，在培养的细胞中未检测到l-DOPA，这可能是由于这些细胞中没有重新合成(6R)-l- red -tetrahydrobiopterin （bh4）。当培养基中加入bh4时，不仅在细胞中检测到大量的左旋多巴，而且在培养基中也检测到大量的左旋多巴。这些发现可能有助于调节细胞移植到大脑后左旋多巴的分泌量。
In the present study, tyrosine hydroxylase (TH; EC 1.14. 16.2) cDNA was transfected into cultured fibroblasts and the production of l-3, 4-dihydroxyphenylalanine (l-DOPA) was determined. Type 2 TH cDNA was transfected into fibroblasts (NRK-49F) derived from the normal rat kidney, and the expression of the TH minigene was screened by immunocytochemical staining and immunoblotting analysis with TH antiserum. Several clones of the NRK transfectants that produce TH molecules were obtained. The expressed TH molecules showed high enzyme activity in a complete assay system in vitro. However, l-DOPA was not detected in the cultured cells due to the possible absence of de novo synthesis of (6R)-l-erythro-tetrahydrobiopterin (BH 4) in these cells. When BH 4 was added to the medium, a large amount of l-DOPA was detected not only in the cells but also in the medium. These findings may aid in regulating the amount of l-DOPA secretion from cells after they are transplanted into the brain.