Inactivation of tautomerase activity of macrophage migration inhibitory factor by sulforaphane: a potential biomarker for anti-inflammatory intervention.
Inactivation of tautomerase activity of macrophage migration inhibitory factor by sulforaphane: a potential biomarker for anti-inflammatory intervention.
复制标题
DOI：
10.1158/1055-9965.epi-11-0279
复制
发表时间：
2011-07
期刊：
影响因子：
--
通讯作者：
Talalay P
中科院分区：
文献类型：
--
作者：
Healy ZR;Liu H;Holtzclaw WD;Talalay P
巨噬细胞迁移抑制因子（Macrophage migration inhibitory factor， MIF）是一种具有酮烯醇互变酶活性的促炎细胞因子，在炎症反应中迅速升高，并在许多慢性疾病中升高。异硫氰酸酯，如西兰花中的萝卜硫素，是非常有效的MIF变异体酶活性灭活剂。因此，一种测定组织和体液中这种活性的简单快速方法可能对评估炎症的严重程度和干预效果有价值。现有的基于甲基l -多巴胺转化为甲基5,6-二羟基吲哚-2-羧酸甲酯的MIF分光光度测定方法在475 nm处的吸收损失缺乏灵敏度。通过降低非酶促反应速率，降低pH至6.2，用Bis-Tris缓冲液取代磷酸盐（催化反应），并转换为微量滴度板格式，检测灵敏度和效率显着提高。异硫氰酸酯对MIF变异体酶失活的结构效价研究表明，萝卜硫素、苄基、正己基和苯乙基异硫氰酸酯对MIF变异体酶失活特别有效。超滤浓缩尿中MIF易定量。该活性包括：(i)在超滤过程中浓缩的热不稳定，萝卜硫素灭活的大分子部分（可能是MIF）；（ii）流动馏分，在过滤过程中具有恒定的活性，热稳定，对萝卜硫素不敏感。人类志愿者服用萝卜硫素前体萝卜硫素几乎完全消除了尿中互变酶活性，并在数小时后恢复。一种简单、快速、定量的MIF变异体酶测定方法已经被开发出来，作为评估炎症严重程度和干预效果的潜在生物标志物。描述了一种改进的测定MIF变异体酶活性的方法及其应用。
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine with keto-enol tautomerase activity, rises rapidly in response to inflammation, and is elevated in many chronic diseases. Isothiocyanates, such as sulforaphane from broccoli, are very potent inactivators of MIF tautomerase activity. A simple rapid method for determining this activity in tissues and body fluids may therefore be valuable for assessing severity of inflammation and efficacy of intervention. Existing spectrophotometric assays of MIF, based on conversion of methyl L-dopachrome to methyl 5,6-dihydroxyindole-2-carboxylate and associated loss of absorption at 475 nm, lack sensitivity. Assay sensitivity and efficiency were markedly improved by reducing the nonenzymatic rate, by lowering pH to 6.2, replacing phosphate (which catalyzes the reaction) with Bis-Tris buffer, and converting to a microtiter plate format. A structure-potency study of MIF tautomerase inactivation by isothiocyanates showed that sulforaphane, benzyl, n-hexyl, and phenethyl isothiocyanates were especially potent. MIF tautomerase could be readily quantified in human urine concentrated by ultrafiltration. This activity comprised: (i) a heat-labile, sulforaphane-inactivated macromolecular fraction (presumably MIF) that was concentrated during ultrafiltration; (ii) a flow-through fraction, with constant activity during filtration, that was heat-stable, and insensitive to sulforaphane. Administration of the sulforaphane precursor glucoraphanin to human volunteers almost completely abolished urinary tautomerase activity, which was recovered over many hours. A simple, rapid, quantitative MIF tautomerase assay has been developed as a potential biomarker for assessing inflammatory severity and effectiveness of intervention. An improved assay for measuring MIF tautomerase activity and its applications are described.