Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells.
Characterization of rabbit liver cytochrome P-450 (laurate omega-1 hydroxylase) synthesized in transformed yeast cells.
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转化酵母细胞中合成的兔肝细胞色素 P-450（月桂酸 omega-1 羟化酶）的表征。
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发表时间：
1988
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通讯作者：
Y. Imai
中科院分区：
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作者：
Y. Imai
构建细胞色素P-450之间的嵌合体(pHP3和pHP2-1)的三个cDNA，并将其插入酵母表达载体pAAH5的醇脱氢酶启动子和终止子区域之间，以形成表达质粒pAH3P2、pAH3E2和pAH3A2。 pAH3P2包含细胞色素P-450(pHP2-1)的完整编码序列，总共490个氨基酸中除了第3、第8、第36和第42个残基之外。在编码NH2末端210和262个氨基酸残基的区域中，pAH3P2的核苷酸序列被替换为细胞色素P-450(pHP3)的核苷酸序列，分别产生pAH3E2和pAH3A2。将三种表达质粒引入酿酒酵母 AH22 细胞，并从转化酵母细胞的微粒体部分中纯化细胞色素 P-450 s（3P2、3E2 和 3A2）。在氧化状态下，两种细胞色素均表现出细胞色素 P-450 的低自旋和高自旋混合型光谱。细胞色素的还原 CO 复合物在 450 nm 处显示 Soret 吸收最大值。当向铁细胞色素 P-450（3P2 和 3E2）中添加月桂酸盐或癸酸盐时，光谱转换为典型的高自旋类型，表明脂肪酸与细胞色素的底物位点结合。另一方面，向铁细胞色素 P-450 (3A2) 添加脂肪酸不会引起光谱变化。在所检测的十二烷基化合物中，只有具有羧基的化学物质才会引起细胞色素 P-450 (3P2) 的光谱转换。（摘要截断为 250 字）
Three cDNAs for chimeras between cytochrome P-450s (pHP3 and pHP2-1) were constructed and inserted between the alcohol dehydrogenase promoter and terminator regions of the yeast expression vector pAAH5 to form expression plasmids, pAH3P2, pAH3E2, and pAH3A2. pAH3P2 contained the entire coding sequence of cytochrome P-450 (pHP2-1) except for the 3rd, the 8th, the 36th, and the 42nd residues of the total of 490 amino acids. Nucleotide sequences of pAH3P2 were replaced with those of cytochrome P-450 (pHP3) in the region coding for the NH2-terminal 210 and 262 amino acid residues to yield pAH3E2 and pAH3A2, respectively. The three expression plasmids were introduced into Saccharomyces cerevisiae AH22 cells and cytochrome P-450 s (3P2, 3E2, and 3A2) were purified from the microsomal fractions of the transformed yeast cells. In the oxidized state either of the cytochromes exhibited a low- and high-spin mixed-type spectrum of cytochrome P-450. The reduced CO complex of the cytochromes showed a Soret absorption maximum at 450 nm. When laurate or caprate was added to ferric cytochrome P-450 s (3P2 and 3E2), the spectrum was converted to that of the typical high-spin type, indicating the binding of the fatty acids to the substrate site of the cytochromes. On the other hand, the addition of the fatty acids to ferric cytochrome P-450 (3A2) induced no spectral change. Only chemicals having a carboxyl group caused such spectral conversion of cytochrome P-450 (3P2) among dodecyl compounds examined.(ABSTRACT TRUNCATED AT 250 WORDS)