Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes
Fluorimetric detection of aldehyde dehydrogenase activity in human blood, saliva, and organ biopsies and kinetic differentiation between class I and class III isozymes
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DOI：
10.1006/abio.1996.9921
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发表时间：
1997-02-01
影响因子：
2.9
通讯作者：
Interewicz, E
中科院分区：
文献类型：
--
作者：
Wierzchowski, J;Wroczynski, P;Interewicz, E
两种高荧光醛，7-甲氧基-1-萘甲醛（MONAL-71）和6-甲氧基-2-萘甲醛（MONAL-62），作为人体组织匀浆和可接近体液中醛脱氢酶（ALDH）活性的指标进行了检查。先前发现这两种化合物是来自红细胞的ALDH和来自人肝脏的纯化I类（胞质）ALDH的优良底物。相比之下，只有MONAL-62，而不是异构体MONAL-71，被人唾液中存在的IH类ALDH氧化。前者与唾液ALDH反应的表观Km为0.24 μ M，反应速率（V-max）接近苯甲醛氧化的速率。苯甲醛对MONAL-62的荧光氧化也有完全竞争性抑制作用。NAD（+）和NADP（+）都可以在该反应中用作氧化剂，具有相当的速率，这是先前针对人III类醛脱氢酶报道的事实。在人肝匀浆（细胞溶质+微粒体部分）中，使用MONAL-71或MONAL-62可轻松检测ALDH活性，比活性分别约为2.5和3.2单位/克蛋白质。低表观K-m值，0.85和
Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicators of the aldehyde dehydrogenase (ALDH) activity in human tissue homogenates and accessible body fluids. Both compounds were previously found to be excellent substrates for the ALDH from erythrocytes and for the purified class I(cytosolic) ALDH from human Liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class IH ALDH present in human saliva. The apparent K-m for the former compound reacting with saliva ALDH is 0.24 mu M, with the reaction rate (V-max) close to that of benzaldehyde oxidation. There is also a fully competitive inhibition of the fluorogenic oxidation of the MONAL-62 by benzaldehyde. Both NAD(+) and NADP(+) can be used as oxidants in this reaction, with comparable rates, a fact previously reported for the human class III aldehyde dehydrogenase. In human liver homogenate (cytosolic + microsomal fraction), the ALDH activity is easily detectable using either MONAL-71 or MONAL-62, with specific activities of approximately 2.5 and 3.2 units per gram of protein, respectively. The low apparent K-m values, 0.85 and