Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members.
Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members.
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DOI：
10.1042/bj3430487
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发表时间：
1999-10
期刊：
影响因子：
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通讯作者：
T. O'connor;L. Ireland;D. J. Harrison;J. Hayes
中科院分区：
文献类型：
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作者：
T. O'connor;L. Ireland;D. J. Harrison;J. Hayes
在大肠杆菌中表达了编码人黄曲霉毒素B(1)醛还原酶（AKR7A2）、醛还原酶（AKR1A1）、醛糖还原酶（AKR1B1）、二氢二醇脱氢酶1 （AKR1C1）和十氯酮还原酶（AKR1C4）的互补DNA克隆。这些醛酮还原酶（AKR）超家族的成员已经从大肠杆菌中纯化为重组蛋白。最近发现的AKR7A2与AKR1同工酶不同，它们能够催化2-羧基苯甲醛的还原。此外，AKR7A2对底物的特异性较窄，其活性仅限于琥珀酸半醛（SSA）、2-硝基苯甲醛、吡啶-2-醛、isatin、1,2-萘醌（1,2- nq）和9,10-菲醌。相比之下，AKR1A1能广泛还原羰基化合物，对SSA、4-羧基苯甲醛、4-硝基苯甲醛、吡啶-3-醛、吡啶-4-醛、4-羟基壬烯醛、苯乙二醛、甲基乙二醛、2,3-己二酮、1,2 - nq、16-酮酮和d-葡萄糖醛酸表现出最高的比活性。AKR7A2和AKR1A1的动力学性质比较表明，两种重组酶对SSA、1,2- nq和16-酮的k(cat)/ k(m)值大致相似。许多作为AKR1A1底物的化合物也可以作为AKR1B1的底物，尽管后者对大多数所研究的芳香族和脂肪族醛的特异性活性明显低于AKR1A1。没有发现AKR1C1和AKR1C4对脂肪醛、芳香醛、醛糖或二羰基具有高还原酶活性。然而，与AKR1A1和AKR1B1不同，AKR1C1和AKR1C4都能催化1-苊酚的氧化，此外，AKR1C4还能氧化二羟基化和三羟基化胆汁酸。针对AKR7A2， AKR1A1, AKR1B1， AKR1C1和AKR1C4的特异性抗体已被用于显示人肝细胞质中所有还原酶的存在；在酒精相关损伤的肝脏中，AKR1B1和AKR1C1的水平显著升高，事实上，AKR1B1仅在酒精性肝病的肝脏中可检测到。脑、心、肾、肝、肺、前列腺、骨骼肌、小肠、脾脏和睾丸提取物的Western blotting显示，AKR7A2存在于所有被检查的器官中，AKR1B1同样广泛分布于人体组织中。然而，这些实验表明，AKR1A1的表达主要局限于脑、肾、肝和小肠。AKR1C家族成员并不像其他还原酶那样广泛表达，AKR1C1仅在肾脏、肝脏和睾丸中被观察到，而AKR1C4仅在肝脏中被发现。由于人类肾脏是AKR的丰富来源，因此对该器官的同工酶进行了进一步的研究。Q-Sepharose上的人肾细胞质阴离子交换层析可以分离AKR1A1， AKR1B1， AKR1C1和AKR7A2，通过底物特异性和Western blotting鉴定。人肾免疫组化表明，AKR7A2在近端和远端曲肾小管中的表达方式与AKR1家族成员相似。此外，AKR7A2和AKR1成员在肾癌细胞中均有表达，提示这两组同工酶可能参与了相关的生理功能。
Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions.