Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue.
Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue.
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DOI：
10.1016/0304-3835(88)90246-7
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发表时间：
1988-09
期刊：
影响因子：
9.7
通讯作者：
D. Smith;L. Martin;R. Wallin
中科院分区：
文献类型：
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作者：
D. Smith;L. Martin;R. Wallin
黄素蛋白DT-黄递酶（EC 1.6.99.2）被认为在身体的防御系统中起重要作用。这种酶已被纯化13，000倍，从接受择期手术的肥胖患者获得的腹部脂肪的胞质级分中回收率为58%。经偶氮二香豆素Sepharose 6 B亲和层析和DEAESephacel阴离子交换层析两步纯化后，酶的纯度达到电泳纯。该酶在SDS-PAGE中表现出32 kDa的单体分子量，并且每32 kDa单体具有1个FAD辅基。FAD辅基似乎牢固地附着在脱辅基蛋白上。该酶还原偶氮染料和醌类，并表现出广泛的底物特异性。该酶具有与从啮齿动物肝脏中纯化的DT-心肌黄酶相似的特性，特别是大鼠肝脏酶。NADH、NADPH和甲萘醌的Km估计值分别为200、140和3.3 μM。三种底物的Vmax依次为762、667和294 μmol/mg·min。药物的抑制常数（Ki）估计分别为10 nM和2.2 μM。当与几种其他组织相比时，腹部脂肪具有最高的DT-黄递酶活性之一（Martin，L.F.，帕特里克，S.D.和Wallin，R.（1987）病态肥胖患者的DT-心肌黄酶。癌症快报，36，341 - 347），但该酶在人类脂肪中的具体作用尚不清楚。
The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Kmvalues for NADH, NADPH and menadione are 200, 140 and 3.3 μM, respectively. Vmaxvalues for these substrates in the same order are 762, 667 and 294 μmol/mg·min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 μM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341 – 347), but the specific role of the enzyme in human fat is unknown.