Furukawa A, Okuyama E, Sumi T, Ichikawa Y
Department of Biochemistry, School of Medicine, Kagawa Medical University, Kita-gun, Japan.
Biol Reprod. 1997 May;56(5):1336-42. doi: 10.1095/biolreprod56.5.1336.
Complementary DNA clones of mRNAs for sheep and goat NADPH-ferredoxin reductases (ferredoxin NADP+ oxidoreductase, EC 1.18.1.2) were isolated by the reverse transcriptase polymerase chain reaction method, and the complete nucleotide sequences of the coding and 3'-flanking regions of these cDNA clones were determined. Comparative analysis using the deduced amino acid sequences of NADPH-ferredoxin reductases clarified the interspecific conservation of the ferredoxin-binding and flavin adenine dinucleotide (FAD)-binding regions, confirming the results reported previously. During this study, we happened to identify an alternatively spliced mRNA that completely lacks exon 3, just adjacent to the FAD-binding region of the sheep NADPH-ferredoxin reductase cDNA clone. In the screening of other alternatively spliced mRNAs of NADPH-ferredoxin reductases derived from several steroidogenic organs, such as adrenocortices, testes, and ovaries of sheep and goats, only one kind of alternatively spliced mRNA as described above was detected in sheep adrenocortices. Then, we constructed Escherichia coli expression systems for these two forms of mRNA and analyzed their enzymatic properties. We found that the ability of the alternatively spliced NADPH-ferredoxin reductase protein to transfer electrons to ferredoxin is completely abolished because FAD binding is inhibited.
通过逆转录酶聚合酶链反应法分离出绵羊和山羊NADPH-铁氧化还原蛋白还原酶（铁氧化还原蛋白NADP+氧化还原酶，EC 1.18.1.2）的mRNA互补DNA克隆，并测定了这些cDNA克隆编码区和3'-侧翼区的完整核苷酸序列。利用NADPH-铁氧化还原蛋白还原酶推导的氨基酸序列进行的比较分析，阐明了铁氧化还原蛋白结合区和黄素腺嘌呤二核苷酸（FAD）结合区的种间保守性，证实了先前报道的结果。在这项研究中，我们偶然发现了一种选择性剪接的mRNA，它完全缺失外显子3，该外显子紧邻绵羊NADPH-铁氧化还原蛋白还原酶cDNA克隆的FAD结合区。在筛选来自绵羊和山羊的肾上腺皮质、睾丸和卵巢等几种类固醇生成器官的NADPH-铁氧化还原蛋白还原酶的其他选择性剪接mRNA时，仅在绵羊肾上腺皮质中检测到一种上述选择性剪接的mRNA。然后，我们为这两种形式的mRNA构建了大肠杆菌表达系统，并分析了它们的酶学性质。我们发现，由于FAD结合受到抑制，选择性剪接的NADPH-铁氧化还原蛋白还原酶蛋白将电子转移到铁氧化还原蛋白的能力完全丧失。
