THE PURIFICATION AND PROPERTIES OF BOVINE PINEAL S‐ADENOSYLMETHIONINE: N‐ACETYLSEROTONIN O‐METHYL TRANSFERASE
THE PURIFICATION AND PROPERTIES OF BOVINE PINEAL S‐ADENOSYLMETHIONINE: N‐ACETYLSEROTONIN O‐METHYL TRANSFERASE
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牛松果体S-腺苷甲硫氨酸N-乙酰血清素O-甲基转移酶的纯化及性质
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发表时间：
1972
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影响因子：
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通讯作者：
Ozand Pt
中科院分区：
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作者：
Karahasanoğlu Am;Ozand Pt
— 牛松果体 S-腺苷甲硫氨酸：N-乙酰血清素 O-甲基转移酶已使用细胞分级、硫酸铵处理、Sephadex G-200 凝胶过滤和阴离子交换色谱法纯化约 2800 倍。已发现该酶是一种聚合物；观察到的最小单位是摩尔。重量。 21,800，其他聚合物的分子量是这个数字的倍数。腺体提取物中聚合物的摩尔数为 83,000、100,000、125,000 和 150,000。重量。比其他人更丰富；它们还表现出更高的比活性。该反应的产物之一，S-腺苷同型半胱氨酸被发现是一种有效的抑制剂，而另一种产物褪黑激素，即使在更高的浓度下也不会抑制牛松果体酶。同型半胱氨酸、半胱氨酸、GSG 和 GSSG 抑制该酶。达到这种效果所需的浓度比S-腺苷高半胱氨酸高100倍。在纯化过程中向培养基中添加 GSH 会导致活性完全丧失。腺苷、同型半胱氨酸和其他硫代化合物几乎没有影响或没有影响。发现该酶被其底物以及某些阴离子激活。在各种有机酸盐中，柠檬酸循环中间体被发现是良好的活化剂；它们的未取代类似物没有那么有效。草酰乙酸盐和碳酸氢盐的活化剂效果最高，并且是由相对较低浓度的这些阴离子（1–5 × 10−3 M）带来的，因此它们的效果被认为是特异性的。草酰乙酸引起的活化程度随着底物浓度的增加而降低，反之亦然。增加底物浓度并不能降低S-腺苷同型半胱氨酸的抑制作用； S-腺苷同型半胱氨酸也抑制草酰乙酸激活酶。这些观察结果已通过酶的变构行为来解释。还研究了各种聚合物的动力学行为。对于 83,000、100,000、125,000 和 150,000 mol 的聚合物，观察到最高的底物和草酰乙酸激活以及最高的 S-腺苷同型半胱氨酸抑制。重量。这些聚合物的草酰乙酸激活酶计算的 S-腺苷甲硫氨酸和 N-乙酰血清素的 Km 值也低于其他聚合物。
— Bovine pineal gland S-adenosylmethionine: N-acetylserotonin O-methyltransferase has been purified about 2800-fold using cell fractionation, ammonium sulphate treatment, Sephadex G-200 gel filtration and anion exchange chromatography. The enzyme has been found to be a polymer; the smallest unit observed had a mol. wt. of 21,800 and the other polymers' molecular weights were multiples of this figure. In the gland extract polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. were more abundant than the others; they showed also higher specific activity. One of the products of the reaction, S-adenosylhomocysteine was found to be a potent inhibitor, whereas the other product, melatonin, did not inhibit the bovine pineal gland enzyme, even at much higher concentrations. Homocysteic acid, cysteic acid, GSG and GSSG inhibited the enzyme. The required concentrations for this effect was 100 times higher than that of S-adenosylhomocysteine. The addition of GSH to the medium during purification led to complete loss of activity. Adenosine, homocysteine and other thio compounds had little or no effect. The enzyme was found to be activated by its substrates and also by certain anions. Among various organic acid salts, citric acid cycle intermediates were found to be good activators; their nonsubstituted analogues were not as effective. The activator effect of oxaloacetate and bicarbonate was the highest, and was brought about by relatively low concentrations of these anions (1–5 × 10−3 M), hence their effect was considered specific. The degree of activation caused by oxaloacetate was decreased by increasing substrate concentrations and vice versa. The S-adenosylhomocysteine inhibition could not be reduced by increasing the substrate concentration; S-adenosylhomocysteine also inhibited the oxaloacetate-activated enzyme. These observations have been explained by the allosteric behaviour of the enzyme. The kinetic behaviour of various polymers was also investigated. The highest substrate and oxaloacetate activation and the highest S-adenosylhomocysteine inhibition was observed for polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. The Km values for S-adenosylmethionine and N-acetylserotonin calculated for the oxaloacetate activated enzyme were also lower for these polymers than others.