Use of catechol O‐methyltransferase for the enzyme radiochemical assay of dopamine
Use of catechol O‐methyltransferase for the enzyme radiochemical assay of dopamine
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儿茶酚 O-甲基转移酶在多巴胺酶放射化学测定中的应用
DOI：
10.1111/j.1471-4159.1973.tb07587.x
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发表时间：
1973
影响因子：
4.7
通讯作者：
L. Iversen
中科院分区：
文献类型：
--
作者：
C. Cuello;R. Hiley;L. Iversen
以前的报告(Saelens等人，1967；Iversen和Jarrot，1970；NIKODIJEVIK等人，1969)描述了甲基转移酶酶苯乙醇胺N-甲基转移酶和邻苯二酚O-甲基转移酶与~(14)Cor~(3+)标记的S-腺苷甲硫氨酸一起用于去甲肾上腺素的酶放射化学测定。按照NIKODIJFXIK et AZ所描述的方法。(1969)用从大鼠肝脏制备的高纯度邻苯二酚甲基转移酶(COMT)测定组织去甲肾上腺素含量。酶反应的产物去甲肾上腺素是通过有机溶剂萃取回收的。然而，这种方法不能应用于同时存在去甲肾上腺素和多巴胺的组织，因为这些胺是COMT的底物，两种胺的甲基化产物将在有机相中回收。本方法用于在去甲肾上腺素或其他生物胺存在的情况下，测定组织提取物中的少量多巴胺。该方法的高灵敏度使其有可能测量非常小的组织样本中的多巴胺。经过一系列的初步研究，我们采用了以下程序：在每次实验开始时新鲜制备反应混合物，它由300pl牛肝COMT在0.5M-磷酸二氢钠缓冲液中组成，pH 7.0，每毫升含有约50 mg蛋白质(制备见下文)；用非放射性S-腺苷蛋氨酸稀释至比活度为600mCi/mmo1或150pl[14C-]甲基S-腺苷蛋氨酸(10pi/ml)(S.A.55mCi/mmo1；放化中心，英格兰，amersham)；50pl含有80 mM-pargyline和10%(v/v)2-巯基乙醇的溶液。在使用前加入325p1的含有30m-氯化镁的1M-Tris碱。为了使最终孵化混合物的pH值达到9.1，这是COMT反应的最佳条件，这一数量的Tris是必要的。在冰冷的200p1微管中加入25微升的这种混合物，然后加入10pl的0.1M-高氯酸(空白)，或10pl在0.1M-高氯酸中含有1.25-12.5 ng多巴胺的标准溶液，或10pl在0.1M-高氯酸中提取的组织样品，除了一些组织样品外，还加入2pl含有5或10 ng多巴胺的水溶液作为内标。组织提取物的制备方法如下。解剖的脑区(0.3-35.0 mg湿重)被放置在体积为1-20的微管中。0.1M-高氯酸，酸量根据预期的多巴胺浓度范围进行调整。用解剖针尖将组织打碎，冷冻解冻，在6000克的微型离心机中离心5-10分钟。取10微升的上清液用于多巴胺的测定。用涡流混合器搅拌孵化混合物和样品，并在37℃孵化15分钟。将试管浸泡在加盐的冰浴中即可停止反应。Whatman 1号色谱纸上有20微升(4x5pl)的斑点。原产地以前是以5pl的3-甲氧基酪胺溶液(10 mg/ml的0.01M-高氯酸)为载体发现的。色谱图以叔戊醇(2-甲基丁烷-2-01)：25-30%甲胺(4：1v/v)下降24~48h展开。在这些条件下，3-甲氧基酪胺和去甲肾上腺素的Rf值有很大的不同，可以得到清晰的分离。3-甲氧基酪胺斑点在紫外灯(254 Nm)下可见，当其产生微弱的紫色荧光时，去甲肾上腺素也可被视作黄色斑点。标记甲氧基酪胺斑点，切下，切碎，放入计数瓶中，在3毫升乙基：浓氨(100：22 V/V)溶液中提取4-18小时。加入12毫升含有0.4%丁基多溴联苯(CIBA)的Triton X-100：甲苯(1：2)混合物后，计算放射性。
PREVIOUS reports (SAELENS et al., 1967; IVERSEN and JARROTT, 1970; NIKODIJEVIK et al., 1969) have described the use of the methyl transferase enzymes phenylethanolamine N-methyltransferase and catechol 0-methyltransferase in conjunction with 14Cor 3H-labelled S-adenosyl methionine for the enzyme radiochemical assay of noradrenaline. In the method described by NIKODIJFXIK et aZ. (1969) the noradrenaline content of tissues was assayed with the use of highly purified catechol 0methyltransferase (COMT) prepared from rat liver. The product of the enzyme reaction, normetanephrine, was recovered by extraction into an organic solvent. This method, however,cannot be applied to tissues in which both noradrenaline and dopamine are present, since these amines are substrates for COMT and the methylated products from both amines would be recovered in the organic phase. The present method was devised for the determination of small amounts of dopamine in tissue extracts, in the presence of noradrenaline or other biogenic amines. The high sensitivity of the method makes it possible to measure dopamine in very small samples of tissue. After a series of preliminary studies we have adopted the following procedure: a reaction mixture is freshly prepared at the start of each experiment; it is composed of 300 pl of bovine liver COMT in 0.5 M-sodium phosphate buffer, pH 7.0, containing about 50 mg of protein per ml (for preparation see below); 150 pl of [3H-]methyl S-adenosyl methionine (100 pCi/ml) (S.A. 7.9 Ci/mmol, The Radiochemical Centre, Amersham, England) diluted with non-radioactive S-adenosyl methionine to a specific activity of 600 mCi/mmol or 150 pl of [14C-]methyl S-adenosyl methionine (10 pCi/ml) (S.A. 55 mCi/mmol; The Radiochemical Centre, Amersham, England); 50 pI of a solution containing 80 mM-pargyline and 10% (v/v) 2-mercaptoethanol. Just before use 325 p1 of 1 M-triS base containing 30 m-magnesium chloride is added. This amount of tris is necessary to bring the pH of the final incubation mix to 9.1, the optimum for the COMT reaction. Twenty-five microlitres of this mixture is added to ice-cold 200 p1 microtubes, and 10 pl of 0.1 M-perchloric acid (blanks), or 10 p1 of standard solutions containing 1.25-12.5 ng of dopamine in 0.1 M-perchloric acid or 10 pl of the tissue samples extracted in 0.1 M-perchloric acid, are then added, in addition to some tissue samples a further 2 pl of an aqueous solution containing 5 or 10 ng of dopamine was added as an internal standard. Tissue extracts are prepared in the following way. Dissected brain areas (0.3-35.0 mg wet wt.) were placed in microtubes with 1-20 vol. of 0.1 M-perchloric acid, the volume of acid being adjusted according to the expected range of dopamine concentrations. The tissue is disrupted with the tip of a dissecting needle, frozen and thawed and centrifuged in a microcentrifuge at 6000 g for 5-10 min. Ten microlitres of the resulting supernatant fluid is taken for dopamine determinations. The incubation mix and samples are agitated with a vortex mixer, and incubated at 37°C for 15 min. The reaction is stopped by immersing the tubes in a salted ice bath. Twenty microlitres (4 x 5 pl) are spotted on Whatman No. 1 chromatographic paper. The origin is previously spotted with 5 pl of a solution of 3-methoxytyramine (10 mg/ml in 0.01 M-perchloric acid) as carrier. The chromatograms are developed by the descending technique for 24-48 h with t-amyl alcohol (2-methylbutan-2-01): 25-30 % methylamine (4: 1 v/v). Under these conditions the RF values for 3-methoxytyramine and normetanephrine are sufficiently different to give a clear separation. The 3-methoxytyramine spot is visualized under a U.V. lamp (254 nm), when it produces a weak violet fluorescence, normetanephrine can also be visualized as a yellow spot. The methoxytyramine spots are marked, cut out, shredded and extracted in counting vials for 4-18 h in 3 ml of a solution of ethano1:concentrated ammonia (100:22 v/v). The radioactivity is counted after adding 12 ml of a Triton X-100: toluene (1:2) mix containing 0.4% butyl PBD (CIBA).