SELECTIVE PARA- OR META-O-METHYLATION WITH CATECHOL O-METHYL TRANSFERASE FROM RAT LIVER
SELECTIVE PARA- OR META-O-METHYLATION WITH CATECHOL O-METHYL TRANSFERASE FROM RAT LIVER
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DOI：
10.1038/202878a0
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发表时间：
1964-01-01
期刊：
影响因子：
64.8
通讯作者：
DEEDS, F
中科院分区：
文献类型：
--
作者：
MASRI, MS;ROBBINS, DJ;DEEDS, F
我们发现，在完整的动物和存活的肝脏切片中，酚类化合物的0-甲基化过程受到芳香环1·2上的侧链和羟化模式的影响。特别有趣的是，3，4，5-三羟基苯甲酸和2，3，4-三羟基苯甲酸分别在对位和间位专一甲基化。然而，对于3，4-二羟基酚酸，甲基化发生在对位或间位·‘。对于儿茶酚，与儿茶酸相比，在完整的动物中只检测到微量的甲基化，而在肝脏切片中没有检测到甲基化；然而，在这两种情况下，广泛的结合是取消儿茶酚构型的明显的主要机制。给完整动物注射焦性没食子酚后，2-0-甲基焦性没食子酸酯的痕迹表明中间羟基发生了选择性的α-甲基化。本报告表明，这些选择性的0-甲基化模式是底物和分离的O-甲基转移酶之间独特关系的反映，因此是由于酶的识别作用或底物的直接影响。酶是根据Axelrod·方法从大鼠肝上清液中制备的，只是省略了最后一步在磷酸钙凝胶上的吸附。反应混合物为2ml透析酶，含15.6 mg蛋白(按Warburg和Christian测定)，0.4mI。M-MgC12，0.4-1米。M磷酸盐缓冲液，pH 7·4，2 mg(-)-S-腺苷-L-碘甲硫氨酸，2 mg底物。最终体积加水至6毫升，在37℃下继续孵育约3小时。如前面所述，用氯仿-乙酸水(2：1：I)下相作为单向的冲洗溶剂，在第二个方向使用20%的氯化钾水溶液(或在碳-14实验的情况下，使用2%的乙酸水溶液)，对酸化孵化混合物的乙醚提取物进行色层分析。使用未标记的SAM，测试了五种底物：没食子酸、3-0-甲基没食子酸、2，3，4-三羟基苯甲酸、3，4-二羟基肉桂酸(咖啡酸)和焦性没食子酸。在第二个实验中，SAM-甲基。UC(1·8FLC.，2 mg)与没食子酸(2 Mg)和邻苯三酚(2 Mg)在酶存在下(1mL.)孵育。未标记的SAM的色谱图表明，在没食子酸的作用下，只发生了p-0-甲基化(即生成了4-0-甲基没食子酸)，而在2，3，4-三羟基苯甲酸的作用下，只发生了m-0-甲基化(形成了3-甲氧基-2，4-二羟基苯甲酸)。另一方面，在3-0-甲基没食子酸和咖啡酸的作用下，p-和m-0-甲基化都发生了。因此，从咖啡酸得到阿魏酸和异阿魏酸，从3-0-甲基没食子酸得到丁香酸和3，4-二甲氧基-5-羟基苯甲酸。与所研究的其他四种底物相比，邻苯三酚的甲基化程度较低。然而，再一次中间羟基的选择性甲基化导致仅2-0-甲基焦性没食子酸的形成是明显的。对邻苯三酚的测定结果与Archer等人报道的结果不一致，后者得到了1-0-甲基邻苯三酚和2，3-二苯三酚.
WE have shown that the course of 0-methylation of phenolic compounds in the intact animal and with surviving liver slices is influenced by the side-chain and the pattern of hydroxylation on the aromatic ring1• 2. Of particular interest was the finding that 3, 4, 5-trihydroxybenzoic (gallic) and 2, 3, 4-trihydroxybenzoic acids were exclusively methylated at the para and meta positions, respectively. With 3, 4-dihydroxyphenolic acids, however, methylation occurred at either the para or meta position•.'With catecholic phenols, as contrasted to catecholic acids, only a trace of methylation was detected in the intact animal, while none was dotected with liver slices; in both cases, however, extensive conjugation was the evident major mechanism for abolishing the catecholic configuration. The trace of 2-0-methyl pyrogallol obtained when the intact animal was given pyrogallol indicated selective a-methylation of the middle hydroxyl group. The present report shows that these selective patterns of 0-methylation are a reflexion of a unique relationship between the substrates and the isolated 0-methyl transferase enzyme and, thus, are due to a discriminating action of the enzyme or a directing influence of the substrates.The enzyme was prepared from rat supernatant liver fraction according to the method of Axelrod•, except that the final step of adsorption on calcium phosphate gel was omitted. The reaction mixture consisted of 2 ml. of the dialysed enzyme containing 15· 6 mg proteinjml.(determined according to Warburg and Christian•), 0· 4 mi. M MgC12, 0· 4 mi. M phosphate buffer pH 7· 4, 2 mg (-)-S-adenosyl-L-methionine iodide (SAM), and 2 mg substrate. The final volume was made up to 6 ml. with water, and incubation was continued for about 3 h at 37 C. Ether extracts of the acidified incubation mixtures were examined chromatographically as previously described2, using the lower phase of chloroform-acetic acidwater (2: 1: I) as tho irrigating solvent in one direction and 20 per cent aqueous potassium chloride (or 2 per cent aqueous acetic acid in the case of the carbon-14 experiment) in the second direction. Using unlabelled SAM, five substrates were tested: gallic, 3-0-methyl gallic, 2, 3, 4-trihydroxybenzoic, and 3, 4-dihydroxycinnamic (caffeic) acids, and pyrogallol. In a second experiment, SAM-methyl. uc (1· 8 fLC., 2 mg) was incubated with gallic acid (2 mg) and with pyrogallol (2 mg) in the presence of the enzyme (1 mi.). The chromatograms of the experiment with unlabelled SAM indicate that, with gallic acid, only p-0-methylation occurred (that is, 4-0-methyl gallic formed), whereas with 2, 3, 4-trihydroxybenzoic acid, only m-0-methylation occurred (3-methoxy-2, 4-dihydroxybenzoic acid formed). On the other hand, with 3-0-methyl gallic acid and with caffeic acid, both p-and m-0-methylation occurred. Thus, both ferulic and isoferulic acids were obtained from caffeic acid, and both syringic and 3, 4-dimethoxy-5-hydroxybevzoic acid were obtained from 3-0-methyl gallic. Pyrogallol was poorly methylated as compared with the other four substrates investigated. However, once again selective methylation of the middle hydroxyl group resulting in the formation of only 2-0-methyl pyrogallol was evident. The results with pyrogallol are at variance with those reported by Archer et al.•, who obtained 1-0-methyl pyrogallol and 2, 3-di-