Synthesis and Release of Biogenic Amines

这些研究都集中在监管方面 酪氨酸羟基酶、色氨酸羟化酶和色氨酸羟基酶的性质 其辅因子四氢生物蝶呤(BH4)在生物合成中的作用 在大脑中释放儿茶酚胺和5-羟色胺。我们有 鉴定人色氨酸羟基酶(TPH)的两个转录本 这是由3‘端开放阅读框上的选择性剪接引起的。 这两个转录本将给出人类TPH的两个亚型 不同的C末端，其中一个包含推定的 CAMP依赖的蛋白激酶(PKA)位点，而另一个， 其中22个氨基酸的长度不会更长。找到了剪接部位 在我们用RT-PCR检测的所有人体组织中都存在 技术，但两个成绩单的比例不同。我们还有 通过对蛋白质的分析，在蛋白质水平上鉴定了两种亚型 抗TPH的人松果体和脑干提取物 抗体。这些结果提供了有关监管机构的知识 5-羟色胺产生机制是通过调节5-羟色胺的表达实现的 TPH。已有研究表明，TPH可被PKA磷酸化 和钙调蛋白依赖性蛋白激酶II(CamPKII)和BE 在14-3-3蛋白存在下被激活。要确定 TPH中的磷酸化位点，我们在大肠杆菌中表达了这两种异构体 和昆虫细胞。在三种电势下构建了几个突变体 可磷酸化的丝氨酸位点：Ser58、Ser260和Ser443。我们发现 Ser58和Ser443在一定程度上被PKA磷酸化 0.35摩尔/亚基的TPH和0.45摩尔/亚基的TPH， 而Ser260不被PKA磷酸化。 CamPKII对这些丝氨酸的磷酸化程度很差，达到0.08 摩尔/亚基TPh。Ser260是CamPKII的最佳站点，它可以是 磷酸化程度为0.06摩尔/TPH亚基。这个 PKA和CamPKII磷酸化的差异可能 揭示了不同监管机制的一些线索 我们还不知道。此外，Ser260可能是 与14-3-3的互动。然而，由于磷酸化较差， 在这个网站上，我们无法显示出显著的互动 在14-3-3和磷酸化的人TPH之间。与 纹状体切片的体外灌流技术，我们已经证明 BH4在调节多巴胺释放方面起着重要的双重作用： 通过其与一氧化氮合酶的辅因子活性，它刺激 一氧化氮(NO)的形成间接刺激 多巴胺的释放。通过其与酪氨酸的辅酶活性 羟基酶，它还直接刺激多巴胺的合成， 其次，它的发布。

These studies have focused on the regulatory properties of tyrosine hydroxylase, tryptophan hydroxylase and their cofactor tetrahydrobiopterin (BH4) in the biosynthesis and release of catecholamine and serotonin in the brain. We have identified two transcripts of human tryptophan hydroxylase (TPH) resulting from an alternative splicing at the 3' open reading frame. The two transcripts would give two isoforms of human TPH with different C termini, one of which contains a putative cAMP-dependent protein kinase (PKA) site, whereas the other one, which is 22-amino acid longer does not. The splicing site was found to exist in all the human tissues we tested by using RT-PCR techniques, but the ratio of the two transcripts varies. We have also identified the two isoforms at the protein level by analyzing protein extracts from human pineal gland and brainstem with anti-TPH antibody. These results provide knowledge about the regulatory mechanism of serotonin production by regulating the expression of TPH. It has been shown that TPH can be phosphorylated by PKA and calmodulin- dependent protein kinase II (CamPKII) and be activated in the presence of 14-3-3 protein. To identify the phosphorylation sites in TPH, we expressed both isoforms in E.coli and insect cells. Several mutants were constructed at three potential phosphorylatable serine sites: Ser58, Ser260 and Ser443. We found that Ser58 and Ser443 were phosphorylated by PKA to the extent of 0.35 mol/subunit of TPH and 0.45 mol/subunit of TPH, respectively, while Ser260 was not phosphorylated by PKA. CamPKII phosphorylated these serines poorly, to an extent of 0.08 mol/subunit TPH. Ser260 is the best site for CamPKII, it can be phosphorylated to an extent of 0.06 mol/subunit of TPH. The difference between the phosphorylation by PKA and CamPKII may reveal some clues to different regulatory mechanisms about which we have not known. In addition, Ser260 is the putative site for interaction with 14-3-3. However, due to the poor phosphorylation of this site, we were not able to show significant interaction between 14-3-3 and phosphorylated human TPH. With the technique of in vitro perfusion of striatal slices, we have shown that BH4 plays an important dual role in regulating dopamine release: through its cofactor activity with nitric oxide synthase, it stimulates the formation of nitric oxide (NO) which indirectly stimulates dopamine release. Through its coenzyme activity with tyrosine hydroxylase, it also stimulates directly dopamine synthesis and, secondarily, its release.