Bhat P V, Poissant L, Wang X L
Hôtel-Dieu de Montréal, Department of Medicine, University of Montréal, Canada.
Biochem Cell Biol. 1996;74(5):695-700. doi: 10.1139/o96-076.
A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1 and ALDH2. The enzymes ALDH1 and ALDH2 were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate--polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1 enzyme had a pI of 7.45 and exhibited a low Km (6.37 microM) for retinal, while the ALDH2 enzyme was found to have very low Km for acetaldehyde (0.98 microM). Based on its kinetic properties, it is suggested that the ALDH1 enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.
一种依赖烟酰胺腺嘌呤二核苷酸（NAD）的酶可催化视黄醛氧化为视黄酸，该酶已从牛肾中纯化至同质。纯化过程中使用的方法包括在DEAE-琼脂糖上进行离子交换色谱、在Affi-gel蓝上进行亲和色谱以及在Mono-Q阴离子交换柱上进行色谱。在Mono-Q柱上，醛脱氢酶（ALDH）分解为两个活性峰，分别命名为ALDH1和ALDH2。ALDH1和ALDH2酶分别纯化了约114倍和65倍。部分纯化的天然酶在Sephacryl S-200 HR上进行凝胶过滤色谱，显示分子量约为108 kDa。纯化后的酶在非变性条件下进行电泳显示为单一蛋白条带。然而，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明在分子量为55、30和22 kDa的区域有三条蛋白条带。两种酶都表现出广泛的底物特异性，可氧化多种脂肪族和芳香族醛。ALDH1酶的等电点为7.45，对视黄醛的Km值较低（6.37 microM），而ALDH2酶对乙醛的Km值非常低（0.98 microM）。基于其动力学特性，推测ALDH1酶可能是牛肾中将视黄醛氧化为视黄酸的主要酶。
