Wierzchowski J, Wroczynski P, Laszuk K, Interewicz E
Department of Physical Chemistry, Faculty of Pharmacy, Medical School, Warsaw, Poland.
Anal Biochem. 1997 Feb 1;245(1):69-78. doi: 10.1006/abio.1996.9921.
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 III ALDH present in human saliva. The apparent Km for the former compound reacting with salvia ALDH is 0.24 microM, with the reaction rate (Vmax) 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 Km values, 0.85 and < 0.03 microM, respectively, together with the inhibition profile by propionic aldehyde (ID50 in the micromolar range) indicate that both compounds are oxidized primarily by the class I ALDH, further confirmed by low activity (0.4 U/g) with NADP+ as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the activity measured with MONAL-71, 0.4 U/g, is much lower than that with MONAL-62 (5.1 U/g with NAD+ and 3.1 U/g with NADP+), the latter being virtually insensitive to 1 mM propionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured selectively with the two fluorogenic substrates described. In all experiments, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mM 4-methylpyrazole, a known inhibitor of the alcohol dehydrogenase, did not change the resultant ALDH activities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed diminished class I ALDH activities (0.7 and 0.03 U/g), but no evidence for class III ALDH induction. The above observations are discussed in relation to the mechanism of detoxication of cyclophosphamide.
研究了两种高荧光醛类化合物，7-甲氧基-1-萘甲醛（MONAL-71）和6-甲氧基-2-萘甲醛（MONAL-62），作为人组织匀浆和可获取体液中醛脱氢酶（ALDH）活性的指示剂。先前发现这两种化合物都是红细胞中ALDH以及人肝脏纯化的I类（胞质）ALDH的优良底物。相比之下，人唾液中存在的III类ALDH仅氧化MONAL-62，而不氧化异构体MONAL-71。前一种化合物与唾液ALDH反应的表观Km为0.24μM，反应速率（Vmax）接近苯甲醛氧化的反应速率。苯甲醛对MONAL-62的荧光氧化也存在完全竞争性抑制。NAD +和NADP +均可作为该反应的氧化剂，反应速率相当，这一事实先前已针对人III类醛脱氢酶进行过报道。在人肝脏匀浆（胞质+微粒体部分）中，使用MONAL-71或MONAL-62均可轻松检测到ALDH活性，每克蛋白质的比活性分别约为2.5和3.2单位。较低的表观Km值分别为0.85和<0.03μM，以及丙醛的抑制曲线（ID50在微摩尔范围内）表明，这两种化合物主要被I类ALDH氧化，以NADP +作为氧化剂时活性较低（0.4 U/g）进一步证实了这一点。相比之下，在主要含有III类ALDH的人胃中，用MONAL-71测得的活性为0.4 U/g，远低于用MONAL-62测得的活性（用NAD +时为5.1 U/g，用NADP +时为3.1 U/g），后者对1 mM丙醛几乎不敏感。因此，在胃匀浆中，可以用所述的两种荧光底物选择性地测量I类和III类ALDH的活性。在所有实验中，醛氧化酶的活性至少比ALDH的活性低10倍。添加5 mM 4-甲基吡唑（一种已知的醇脱氢酶抑制剂）不会使所得的ALDH活性变化超过10%，表明前一种酶没有干扰。对两个肝肿瘤样本的初步筛查显示I类ALDH活性降低（0.7和0.03 U/g），但没有证据表明III类ALDH被诱导。结合环磷酰胺的解毒机制对上述观察结果进行了讨论。
