Yu Y, Yamasaki H, Kita K, Takamiya S
Department of Parasitology, Juntendo University, School of Medicine, Tokyo, Japan.
Arch Biochem Biophys. 1996 Apr 1;328(1):165-72. doi: 10.1006/abbi.1996.0157.
A b5-type cytochrome was extracted from Ascaris suum muscle at pH 4.5 with 0.3% aluminum sulfate and purified by ammonium sulfate fractionation, ion-exchange chromatography on CM-500 Cellulofine, and gel filtration on Sephadex G-75. The hemoprotein displayed a typical absorption spectrum of cytochrome b with a midpoint redox potential of 78 mV. The N-terminal amino acid sequence was determined and revealed the N-terminus to be highly homologous to the heme-binding domain of vertebrate cytochrome b5. Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encoding A. suum cytochrome b5 was isolated from the lambda ZAP II cDNA library. The entire nucleotide sequence of 563 bases comprised an open reading frame of 339 bases encoding a precursor protein of 112 amino acid residues. The purified cytochrome B5 was predicted to contain 82 amino acids with a molecular mass of 9141 Da, matching the 9140 Da obtained from electrospray ionization mass spectometry, and to lack of membrane-anchor domain at the C-terminus. In contrast, an N-terminal extension of 30 amino acids, characteristic of signal peptides, was apparent. Immunoblots revealed the presence of an A. suum cytochrome b5 of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochrome b5 possessing a presequence.
在pH 4.5条件下，用0.3%的硫酸铝从猪蛔虫肌肉中提取出一种b5型细胞色素，然后通过硫酸铵分级分离、在CM - 500纤维素上进行离子交换色谱以及在Sephadex G - 75上进行凝胶过滤进行纯化。该血红蛋白显示出细胞色素b的典型吸收光谱，其氧化还原电位中点为78 mV。测定了其N端氨基酸序列，结果表明N端与脊椎动物细胞色素b5的血红素结合结构域高度同源。利用基于纯化蛋白氨基酸序列合成的寡核苷酸探针，从λZAP II cDNA文库中分离出编码猪蛔虫细胞色素b5的cDNA克隆。563个碱基的完整核苷酸序列包含一个339个碱基的开放阅读框，编码一个112个氨基酸残基的前体蛋白。预测纯化的细胞色素B5含有82个氨基酸，分子量为9141 Da，与电喷雾电离质谱法测得的9140 Da相符，且C端缺乏膜锚定结构域。相反，明显存在一个30个氨基酸的N端延伸，这是信号肽的特征。免疫印迹显示存在一个82个氨基酸的猪蛔虫细胞色素b5，但没有N端延伸的蛋白。这些结果表明存在一种具有前导序列的新型细胞色素b5。
