Wang C, Bammler T K, Guo Y, Kelly E J, Eaton D L
National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Dr., HFT-100, Jefferson, Arkansas 72079. Roosevelt Way NE, 100, Seattle, Washington 981.
Toxicol Sci. 2000 Jul;56(1):26-36. doi: 10.1093/toxsci/56.1.26.
Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.
小鼠对霉菌毒素黄曲霉毒素B1（AFB1）的致癌作用具有抗性，因为它们组成性地表达一种α类谷胱甘肽S-转移酶（mGSTA3-3），该酶对黄曲霉毒素B1-8,9-环氧化物（AFBO）具有高活性（约200,000 pmol/分钟/毫克）。大鼠不组成性表达具有高AFBO结合活性的谷胱甘肽S-转移酶，并且对AFB1诱导的肝癌发生敏感。组成性表达的人肝α类谷胱甘肽S-转移酶（hGSTA1-1和hGSTA2-2）几乎没有或没有AFBO解毒活性（<2 pmol/分钟/毫克）。最近，我们发现非人灵长类动物食蟹猴（Mf）对AFBO表现出显著的（约300 pmol/分钟/毫克）组成性肝谷胱甘肽S-转移酶活性。为了确定哪种特定的谷胱甘肽S-转移酶同工酶负责这种活性，从肝组织中纯化了Mf谷胱甘肽S-转移酶并进行了表征，并且通过逆转录酶偶联聚合酶链反应（RT-PCR）克隆了Mf μ类谷胱甘肽S-转移酶cDNA。通过谷胱甘肽琼脂糖（GSHA）亲和色谱法纯化得到一种蛋白质GSHA-GST，与其他含谷胱甘肽S-转移酶的峰相比，它表现出相对较高的AFBO结合活性（239 pmol/分钟/毫克）。蛋白质免疫印迹和酶活性分析表明GSHA-GST属于μ类。通过RT-PCR产生了两种不同的μ类谷胱甘肽S-转移酶cDNA，mfaGSTM1（GenBank登录号#AF200709）和mfaGSTM2（GenBank登录号#AF200710）。cDNA推导的氨基酸序列分析表明，mfaGSTM1和mfaGSTM2分别与人μ类谷胱甘肽S-转移酶hGSTM4和hGSTM2具有97％和96％的同源性。与没有可检测到AFBO结合活性的重组mfaGSTM1-1相反，mfaGSTM2-2以333 pmol/分钟/毫克的活性表现出这种活性。纯化的蛋白质GSHA-GST和重组mfaGSTM2-2的外消旋体和内消旋体AFBO以及1-氯-2,4-二硝基苯的活性谱表明它们是两种不同的酶。我们的结果表明，与啮齿动物相反，μ类谷胱甘肽S-转移酶负责食蟹猴肝脏中大部分的AFBO结合活性。
