Rat liver cholesterol 7 alpha-hydroxylase. Pretranslational regulation for circadian rhythm.
Rat liver cholesterol 7 alpha-hydroxylase. Pretranslational regulation for circadian rhythm.
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DOI：
10.1016/s0021-9258(19)38775-7
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发表时间：
1990-06
期刊：
影响因子：
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通讯作者：
M. Noshiro;M. Nishimoto;K. Okuda
中科院分区：
文献类型：
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作者：
M. Noshiro;M. Nishimoto;K. Okuda
编码胆固醇7 α-单加氧酶（EC 1.14.13.17）的完整cDNA，其已通过使用酶的特异性抗体从大鼠肝cDNA文库中分离（Noshiro，M.，Nishimoto，M.，和Okuda，K. 04 The Dog（1989）257，97-100）完全测序。该cDNA含有一个1，509碱基对的开放阅读框，编码503个氨基酸残基（Mr = 56，880）和一个异常长的3 '非翻译区，全长3，545碱基对，富含AT序列。预测的氨基酸序列显示出与其他测序的细胞色素P-450的相似性小于30%，表明7 α-羟化酶构成了细胞色素P-450的新家族。富含AT的区域通常含有ATTTA基序、5 '-AAT-3'或5 '-TAA-3'三核苷酸，据报道这些三核苷酸参与快速降解mRNA。采用特异性抗体和cDNA作为探针，在一天中不同时间制备的大鼠肝脏上研究了三种因子（即酶蛋白、mRNA和酶活性）水平的日变化。在正常动物中，所有三个因素均在晚上10：00达到最高水平，上午10：00达到最低水平，未观察到显著的性别差异。在上午10：00时，考来烯胺喂养使所有三个因子都接近正常大鼠的最大水平，但在晚上10：00时没有显示出显著的增加。相反，饥饿在上午10：00时或晚上10：00时显著降低了所有三个因子，同时保持昼夜变化。mRNA水平与酶活性和蛋白质水平的良好相关性表明，翻译前调节很可能是7 α-羟化酶昼夜节律的机制。蛋白质含量和mRNA水平的显著昼夜波动也表明它们的快速周转。mRNA的短半衰期可能与快速降解mRNA的特征性mRNA的3 '-非翻译区的结构相关，即基序、AUUUA的丰度以及二级结构的单链区域中5'-AAU-3'或5'-UAA-3'三核苷酸的存在。
A complete cDNA encoding cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) which had been isolated from rat liver cDNA libraries by using specific antibodies to the enzyme (Noshiro, M., Nishimoto, M., and Okuda, K. (1989) FEBS Lett. 257, 97-100) was totally sequenced. The cDNA contained a 1,509-base pair open reading frame encoding 503 amino acid residues (Mr = 56,880) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3,545 base pairs. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s indicating that the 7 alpha-hydroxylase constitutes a novel family of cytochrome P-450. The AT-rich region often contained ATTTA motifs, 5'-AAT-3' or 5'-TAA-3' trinucleotides which were reported to be involved in rapidly degrading mRNA. Employing the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors, i.e. enzyme protein, mRNA, and enzyme activity, was studied on rat livers prepared at various times of the day. In normal animals, all three factors exhibited maximum level at 10:00 p.m. and minimum at 10:00 a.m. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10:00 a.m. close to the maximum levels of the normal rats, but did not show a significant increase at 10:00 p.m. On the contrary, starvation markedly decreased all three factors either at 10:00 a.m. or at 10:00 p.m., while maintaining still the diurnal variation. A good correlation of the levels of mRNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 alpha-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of mRNA also indicates their rapid turnover. The short half-life of mRNA could be correlated with the structure of the 3'-untranslated region of the mRNA characteristic of rapidly degrading mRNA, i.e. abundance of motif, AUUUA, and existence of 5'-AAU-3' or 5'-UAA-3' trinucleotides in single-stranded regions of the secondary structure.