Imamura Y, Koga T, Otagiri M, Satoh K, Hara A
Faculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi, Japan.
Biol Pharm Bull. 1998 Jan;21(1):76-8. doi: 10.1248/bpb.21.76.
Six peptides were obtained by the digestion of carbonyl reductase purified from rabbit liver. The amino acid sequences of the six peptides were virtually identical to the corresponding regions in amino acid sequences deduced from two cloned carbonyl reductase genes (RCBR5 and RCBR6). However, there was a difference of one amino acid residue between the sequences of peptides from the purified enzyme and the corresponding region in the amino acid sequences deduced from the two cDNAs. The purified carbonyl reductase was confirmed to exhibit no reactivity towards menadione, even though the transient expression of the two cDNA for rabbit liver carbonyl reductase has been reported to cause a marked increase of menadione reductase activity in COS7 cells. The enzyme purified from rabbit liver was inactivated by thiol-specific reagents, 5,5'-dithiobis(2-nitrobenzoic acid) and sodium tetrathionate, suggesting that menadione probably interacts with the functional cysteine residue(s), and cannot serve as a substrate of the purified enzyme. Based on these results, it is concluded that the carbonyl reductase purified from rabbit liver is not the product of cloned carbonyl reductase gene (RCBR5 or RCBR6).
通过对从兔肝中纯化得到的羰基还原酶进行消化，获得了六种肽段。这六种肽段的氨基酸序列与从两个克隆的羰基还原酶基因（RCBR5和RCBR6）推导的氨基酸序列中的相应区域几乎完全相同。然而，纯化酶的肽段序列与从两个cDNA推导的氨基酸序列中的相应区域之间存在一个氨基酸残基的差异。尽管据报道兔肝羰基还原酶的两个cDNA的瞬时表达会导致COS7细胞中维生素K3还原酶活性显著增加，但纯化的羰基还原酶被证实对维生素K3没有反应活性。从兔肝中纯化的酶被硫醇特异性试剂5,5'-二硫代双（2-硝基苯甲酸）和连四硫酸钠灭活，这表明维生素K3可能与功能性半胱氨酸残基相互作用，并且不能作为纯化酶的底物。基于这些结果，可以得出结论，从兔肝中纯化的羰基还原酶不是克隆的羰基还原酶基因（RCBR5或RCBR6）的产物。
