Koshizaka T, Nishikimi M, Ozawa T, Yagi K
Institute of Applied Biochemistry, Yagi Memorial Park, Gifu, Japan.
J Biol Chem. 1988 Feb 5;263(4):1619-21.
L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.
L-古洛糖酸-γ-内酯氧化酶是微粒体黄素酶之一，在许多动物体内催化L-抗坏血酸生物合成的最后一步；然而，在易患坏血病的动物如人类、灵长类动物和豚鼠中却不存在这种酶。通过使用针对该酶的抗体筛选λgt11载体中的大鼠肝脏cDNA表达文库，分离出了该酶的cDNA克隆。该cDNA克隆包含2120个核苷酸，一个1320个核苷酸的开放阅读框，编码440个氨基酸的蛋白质，分子量为50,605。从大鼠肝脏中分离出的该酶的氨基末端序列（第1-33位氨基酸残基）与推导的氨基酸序列的相应部分完全一致。推导氨基酸组成与该酶氨基酸分析结果的一致性进一步证实了该cDNA克隆的同一性。对推导氨基酸序列的亲水性分析揭示了几个疏水区域，表明它们将蛋白质锚定在微粒体膜中。推导的氨基酸序列与其他八种黄素酶的黄素结合区域没有明显的同源性。
