Evaluation for adverse reaction of drugs using metabolic parameters

硝酸甘油（GTN）已被用作治疗心绞痛的首选药物。研究表明，一些谷胱甘肽s -转移酶（GSTs）催化了从GTN到硝酸甘油（GDNs）的代谢转化。在这项研究中，我们检测了GSTs对GTN的底物特异性。分别从猪、人和Caco-2细胞的肝脏和粘膜中分离出α和mu GSTs。Mu GSTs对GTN的降解具有时间依赖性，其形成1,3- gdn的比例（1,2- gdn /1,3 - gdn）为0.61，而α gst的形成量是不同水解部分硝基的两倍。我们发现了一种参与吲哚美辛酰胺键水解的酶，并对其进行了部分表征及其底物特异性。纯化后的酶能有效水解吲哚美辛中的酰胺键，但不能水解羧酸酯酶的典型底物-乙酸萘酯和对硝基苯基乙酸酯中的酰胺键。吲哚美辛的Michaelis常数和最大流速值分别为67 mM和9 nmol/mg蛋白/min。这种酶被称为ES-male。猪、兔和人肝脏的吲哚美辛水解活性较高，大鼠和小鼠肝脏的吲哚美辛水解活性较低。纯化了从大鼠肝微粒体中分离得到的两个羧酸酯酶，其pl值分别为6.0和6.2。这两种同工酶在底物特异性上有显著差异。从氨基酸序列分析结果来看，羧酸酯酶pl 6.0和6.2分别与水解酶A和水解酶B相同。羧酯酶pl 6.2能有效水解Pranlukast，而pl 6.0酶则不能，人与大鼠对Pranlukast的代谢差异可以用羧酯酶的底物特异性来解释。

Nitroglycerin (GTN) has been used as the drug of choice in the treatment of angina pectoris. It has been shown that some glutathione S-transferases (GSTs) catalyze the metabolic conversion from GTN to glyceryl dinitrates (GDNs). In this study, we examined the substrate specificity of GSTs for GTN.Alpha and mu GSTs were isolated from the liver and mucosa from pig, human and Caco-2 cells. Mu GSTs degraded GTN time-dependently and formed 1,3-GDN in preference to 1,2-GDN at ratio (1,2-GDN/1,3GDN) of 0.61, whereas alpha GSTs formed twice as much as different hydrolyzing portions of the nitrogroups.We found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in a-naphthylacetate and p-nitrophenylacetate which are typical substrate for carboxylestrase. The Michaelis constant and maximum velocity values for indomethacin were 67 mM and 9 nmol/mg protein/min, respectively. This enzyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver, but not in those form rat and mouse. Two carboxylesteases with pl 6.0 and 6.2 derived from rat liver microsomes were purified. The two isozymes were remarkably different in substrate specificity. Carboxylesterases pl 6.0 and 6.2 are identical to the enzymes referred to as hydrolase A and B,respectively, from the results of amino acid sequence analyzes. Pranlukast was effectively hydrolyzed by caroxylesterase pl 6.2 but not by the pl 6.0 enzyme and the difference in the pranlukast metabolism between the human and the rat could be explained by the substrate specificity of carboxylesteases.