Activation of tryptophan hydroxylase from slices of rat brain stem incubates with N6, 02'-dibutyryl adenosine-3':5'-cyclic monophosphate.
Activation of tryptophan hydroxylase from slices of rat brain stem incubates with N6, 02'-dibutyryl adenosine-3':5'-cyclic monophosphate.
复制标题
大鼠脑干切片中色氨酸羟化酶的激活与 N6, 02-二丁酰腺苷-3:5-环单磷酸一起孵育。
DOI：
10.1016/0006-2952(80)90398-6
复制
发表时间：
1980
影响因子：
5.8
通讯作者：
M. Boadle
中科院分区：
文献类型：
--
作者：
M. Boadle
最近对色氨酸羟化酶（--5-羟色胺 (5-HT) 合成中的限速酶）的体外研究表明，在磷酸化条件下酶活性会增加 (I-3)，并且当反应介质中包含钙（而不是腺苷 3': 5'-环单磷酸 (CAMP)）以及 ATP 和镁时，酶活性的增加会进一步增强 (1, 2)。这些观察结果表明，当从在高钾钠中去极化的脑干切片制备酶时，观察到色氨酸羟化酶活性的钙依赖性增加的分子机制(4, 5)，以及完整神经组织响应电刺激而增强的 5-HT 合成 (6-8)，并且也是钙依赖性的 (9)。根据该方案，在去极化期间进入神经的钙触发色氨酸羟化酶或激活蛋白的钙依赖性磷酸化，这反过来又产生酶的激活和/或刺激，从而导致色氨酸向5-羟基色氨酸(5-HTP)的转化增强。这些对酶粗制剂的实验似乎排除了环核苷酸在色氨酸羟化酶活性调节中的作用，这与密切相关的酶酪氨酸羟化酶形成鲜明对比 (10)。因此，令人惊讶地发现，当从与N6，02'-二丁酰腺苷-3'：5'-环单磷酸（二丁酰CAMP）一起温育的脑干切片制备时，色氨酸羟化酶被激活。这一发现是本报告的主题。本研究中用于脑干切片（间脑、中脑、延髓）的制备和孵育的程序，用于从低速上清液部分中的切片制剂中分离酶，以及用于色氨酸羟化酶测定的程序均已在其他地方详细描述（4, 5）。在本实验中，每个脑干的切片在 25'in 5.0 ml 的添加了不同浓度的二丁酰 CAMP (Sigma Chemical Co.) 的含氧培养基在 10 分钟的孵育期结束时，通过离心将每个切片的脑干从孵育培养基中分离出来，并用作色氨酸羟化酶的低速上清液制剂的来源，在 200 PM L-色氨酸和次优浓度 (50 I) 的还原人工蝶呤辅因子 S-甲基-5 的存在下进行测定。 6, 7, 8-四氢蝶呤（6-MPH4，Calbiochem Behring Corp.）使用 Friedman 等人 (11) 的方法进行修改，如其他地方所述 (4, 5)。结果以每毫克蛋白质每分钟形成的皮摩尔 5-HTP 表示，并且是来自至少三个单独脑干制剂的值的平均值。
Recent in vitro studies on tryptophan hydroxylase, the rate-limiting enzyme in--5-hydroxytryptamine(5-HT) synthesis, have revealed that enzyme activity is increased under phosphorylating conditions (I-3) and that this increase in enzyme activity is further enhanced when calcium, but not adenosine 3': 5'-cyclic monophosphate (CAMP) is included in the reaction medium together with the ATP and magnesium (1, 2). These observations suggest a molecular mechanism for the calcium-dependent increase in tryptophan hydroxylase activity observed when enzyme is prepared from slices of brain stem depolarized in a high potassium odium(4, 5) and for the enhanced synthesis of 5-HT which occurs in intact nervous tissue in response to electrical stimulation (6-8) and which is also calcium dependent (9). According to this scheme, calcium which enters the nerve during depolarization triggers a calciumdependent phosphorylation of tryptnnhan hydroxylase, or an activator protein, and this, in turn, produces an activation and/or stimulation of the enzyme which results in the enhanced conversion of tryptophan to 5-hydroxytryptophan(5-HTP). These experiments on crude preparations of enzyme appear to exclude a role for cyclic nucleotides in the regulation of tryptophan hydroxylase activity, in marked contrast to the closely related enzyme, tyrosine hydroxylase (10). It was therefore surprising to discover that tryptophan hydroxylase was activated when prepared from brain stem slices which had been incubated with N6, 02'-dibutyryl adenosine-3': 5'-cyclic monophosphate (dibutyryl CAMP). This finding is the subject of the present report.The procedures used in this study for the preparation and incubation of the slices of brain stem (diencephalon, midbrain, medulla ponsf, for the isolation of the enzyme from the slice preparation in a low speed supernatant fraction, and for the tryptophan hydroxylase assay have all been described in detail elsewhere (4, 5), In the present experiments the slices of each brain stem were incubated at 25'in 5.0 ml of oxygenated medium to which different concentrations of dibutyryl CAMP (Sigma Chemical Co.) were added. At the end of a ten minute incubation period each sliced brain stem was separated from the incubation medium by centrifugation and used as the source of the low speed supernatant preparation of tryptophan hydroxylase. Enzyme was assayed in the presence of 200 PM L-tryptophan and a suboptimal concentration (50$ I) of reduced artificial pterin cofactor, S-methyl-5, 6, 7, 8-tetrahydropterin(6-MPH4, Calbiochem Behring Corp.) using a modification of the method of Friedman et al.(11) as described elsewhere (4, 5). The activity of each brain stem enzyme preparation was assayed in sextuplet. Results are expressed in pmoles 5-HTP formed per mg protein per minute and are the means? SEM of values from at least three separate brain stem preparations.