The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction
The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction
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DOI：
10.1007/bf03402210
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发表时间：
1996-01-01
影响因子：
5.7
通讯作者：
Rorsman, H
中科院分区：
文献类型：
--
作者：
Rosengren, E;Bucala, R;Rorsman, H
背景资料：最近对黑色素生物合成的研究已经发现了一种不寻常的酶活性，其将2-羧基-2，3-二氢吲哚-5，6-醌（多巴色素）的非天然存在的D-异构体转化为5，6-二羟基吲哚-2-羧酸（DHICA）。本调查的目的是分离和表征的酶催化这种tautomerization reaction.Materials和方法：我们进行了组织调查后，D-多巴色素互变异构酶活性，10牛晶状体均质化，并作为酶的来源。通过高速离心获得可溶性级分，并在苯基琼脂糖凝胶、Mono S阳离子交换和Superdex凝胶过滤上进行连续的FPLC色谱。结果：纯化的D-多巴色素转化为DHICA的催化蛋白纯度为14%，经SDS-PAGE分析其分子量为12 kD。对该蛋白的前27个氨基酸残基进行测序，发现其与牛巨噬细胞移动抑制因子（MIF）的氨基酸残基相同。天然MIF的催化活性通过纯化的重组人MIF的研究证实，其显示出相同的互变异构酶活性。虽然L-多巴色素不是这种反应的底物，甲酯的L-和D-异构体被发现是更好的底物MIF比D-多巴色素。结论：MTF最近被描述为垂体前叶激素和释放的免疫细胞刺激低浓度的糖皮质激素。一旦被分泌，MIF起控制或反调节糖皮质激素对免疫系统的免疫抑制作用的作用。虽然测试的底物，D-多巴色素，不自然发生，观察到MIF具有互变异构酶活性表明，MIF可能介导其生物学效应的酶促反应。这些数据还为设计MIF的小分子药理学抑制剂提供了一种潜在的方法，这些抑制剂可以调节其在体内的强效免疫调节作用。
Background: Recent studies of melanin biosynthesis have uncovered an unusual enzymatic activity which converts the non-naturally occurring D-isomer of 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present investigation was to isolate and characterize the enzyme catalyzing this tautomerization reaction.Materials and Methods: After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing.Results: The protein catalyzing the conversion of D-dopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIF was confirmed by studies of purified recombinant human MIF, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the L- and D-isomers were found to be better substrates for MIF than D-dopachrome.Conclusions: MTF has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.