Ohtomi M, Sasaki M, Deguchi T
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
Eur J Biochem. 1989 Nov 6;185(2):253-61. doi: 10.1111/j.1432-1033.1989.tb15110.x.
A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.
用本实验室最近从鸡肝脏中分离得到的经32P标记的芳胺N - 乙酰转移酶cDNA，对从夜间获取的鸡松果体富含多聚腺苷酸（poly(A)）的RNA构建的cDNA文库进行筛选。分离出两个与肝脏cDNA发生交叉杂交的阳性克隆（p - NAT - 3和p - NAT - 10）。在高严谨度条件下，这两个cDNA彼此不发生交叉杂交，表明它们对应不同的mRNA。当将这些cDNA插入到受猿猴病毒40早期启动子控制的表达载体pcDL1中，并导入中国仓鼠卵巢细胞时，两个cDNA在转染细胞中均表达出芳胺N - 乙酰转移酶活性。测定了cDNA的核苷酸序列，并由此推导氨基酸序列。两个cDNA均编码290个氨基酸。p - NAT - 3、p - NAT - 10与肝脏N - 乙酰转移酶之间的氨基酸序列相似性约为60%。富含多聚腺苷酸RNA印迹杂交分析表明，p - NAT - 3 cDNA与来自脑、肠道的富含多聚腺苷酸RNA以及强度较弱的来自脾脏、肝脏和肾脏的RNA检测到一条2.2 kb的条带，而p - NAT - 10 cDNA仅与来自肾脏的富含多聚腺苷酸RNA杂交。两个cDNA均未检测到与松果体富含多聚腺苷酸RNA的任何杂交条带，提示其含量较低。基因组Southern印迹杂交分析表明，p - NAT - 3、p - NAT - 10和肝脏N - 乙酰转移酶由单独的单个基因编码。将转染细胞中表达的酶的特性与来自松果体、脑和肾脏的N - 乙酰转移酶进行比较。在DEAE - 纤维素柱上，肾脏和p - NAT - 10酶出现在流出组分中，而脑和p - NAT - 3酶在0.08 M NaCl浓度下通过梯度洗脱从柱上洗脱下来。白天获取的松果体上清液在流出组分和0.08 M NaCl浓度下的洗脱组分中出现两个峰。以对乙氧基苯胺、2 - 氨基芴、色胺和苯乙胺作为底物检测这些酶的底物特异性。所有酶对芳胺的偏好高于芳烷基胺，表明p - NAT - 3和p - NAT - 10 cDNA均编码芳胺N - 乙酰转移酶。
