Sanny C G, Rymas K
Department of Biochemistry/Microbiology, Oklahoma State University College of Osteopathic Medicine, Tulsa 74107.
Alcohol Clin Exp Res. 1993 Oct;17(5):982-7. doi: 10.1111/j.1530-0277.1993.tb05652.x.
Methods for analysis of aldehyde dehydrogenase isoenzymes using high-performance (pressure) liquid chromatography (HPLC) were used to determine in vivo effects of disulfiram and cyanamide on canine liver aldehyde dehydrogenase (ALDH) isoenzymes. Liver ALDH isoenzymes from control and disulfiram- or cyanamide-treated dogs were separated by ion-exchange HPLC, and enzyme activity was detected using a postcolumn reactor. Two major peaks of ALDH activity (peaks I and II) were detected. Varying the composition of the reaction column reagents resulted in alterations in the elution profiles consistent with the kinetic properties of individual isoenzymes (i.e., ALDH IB in peak I and ALDH IIB in peak II), including estimates of the Km for acetaldehyde and the effects of magnesium ions on ALDH activity. Disulfiram treatment decreased both peaks depending on disulfiram dose and length of treatment, with peak I being more sensitive to inactivation than peak II. Reagents containing MgCl2 (1 mM) decreased peak I and increased peak II compared with EDTA (1 mM) for samples from both control and disulfiram-treated animals. These data are consistent with the assignment of the disulfiram-sensitive isoenzyme (ALDH IB) to peak I and the isoenzyme stimulated by magnesium ions (ALDH IIB) to peak II. In vivo cyanamide treatment produced similar decreases in both peaks to a maximum decrease of approximately 30% of control depending on cyanamide dose. Peak I, however, was more sensitive than peak II to in vitro inactivation by cyanamide, which suggests that cytosolic ALDH in the dog (in contrast to other mammals) is more sensitive to inactivation than mitochondrial ALDH.
采用高效（压力）液相色谱法（HPLC）分析醛脱氢酶同工酶的方法，用于确定双硫仑和氨基氰对犬肝脏醛脱氢酶（ALDH）同工酶的体内作用。通过离子交换HPLC分离对照犬以及经双硫仑或氨基氰处理的犬的肝脏ALDH同工酶，并使用柱后反应器检测酶活性。检测到两个主要的ALDH活性峰（峰I和峰II）。改变反应柱试剂的组成导致洗脱图谱发生变化，这与各个同工酶的动力学特性一致（即峰I中的ALDH IB和峰II中的ALDH IIB），包括对乙醛Km值的估计以及镁离子对ALDH活性的影响。双硫仑处理会使两个峰均降低，降低程度取决于双硫仑剂量和处理时间，峰I比峰II对失活更敏感。对于来自对照和双硫仑处理动物的样品，与含有1 mM EDTA的试剂相比，含有1 mM MgCl2的试剂会使峰I降低，峰II升高。这些数据与双硫仑敏感同工酶（ALDH IB）归属于峰I以及镁离子刺激的同工酶（ALDH IIB）归属于峰II的结果一致。体内氨基氰处理会使两个峰出现类似程度的降低，根据氨基氰剂量，最大降低幅度约为对照的30%。然而，峰I比峰II对氨基氰的体外失活更敏感，这表明犬的胞质ALDH（与其他哺乳动物不同）比线粒体ALDH对失活更敏感。
