Shen S, Strobel H W
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston 77225, USA.
Arch Biochem Biophys. 1995 Jun 20;320(1):162-9. doi: 10.1006/abbi.1995.1355.
Several positively charged amino acid residues in cytochrome P450 have been shown to be involved in the electrostatic association with NADPH-cytochrome P450 reductase. For cytochrome P4501A1, five regions were proposed as the putative binding sites for the reductase (Shen, S., and Strobel, H. W. (1993) Arch. Biochem. Biophys. 304, 257-265). To elucidate the specific roles of each of these amino acid residues, five anti-peptide antibodies defined as 1A, 4A, 5A, 6A, and 7A were generated against these regions containing 8-13 amino acids and were affinity-purified using a peptide-Sepharose 4B column. Analysis by enzyme-linked immunosorbent assay and protein immunoblot techniques demonstrated that three of five anti-peptide antibodies have specific binding to the peptides as well as to cytochrome P4501A1. Incubation of the various anti-peptide antibodies with cytochrome P450 followed by reconstitution with reductase, lipid, and NADPH resulted in significant inhibition of P450 activity for antibodies 5A and 6A, but not for 1A, 4A, or 7A. Antibody 5A also exhibited inhibition of P450 activity supported by cumene hydroperoxide, though the inhibition was 45 to 30% less than the inhibition of reductase-supported activity at each of the increasing concentrations of antibody. Kinetic studies with antibody 5A revealed no change in the Km for the substrate ethoxycoumarin, but rather a dramatic effect on the Vmax of the cytochrome P4501A1 system whether reconstituted with reductase or supported by cumene hydroperoxide. Characterization of the effects of antibody 5A on cytochrome P4501A1 suggested that the binding of antibody 5A to P4501A1 may change the binding of P4501A1 with reductase. Furthermore, the binding of 5A to cytochrome P4501A1 also lowered the Vmax of the P450. These results are consistent with the roles for the regions of cytochrome P4501A1 from amino acid residues 269 to 281 (peptide 5) and 454 to 463 (peptide 6) in cytochrome P4501A1 hydroxylation activity.
细胞色素P450中几个带正电荷的氨基酸残基已被证明参与了与NADPH - 细胞色素P450还原酶的静电结合。对于细胞色素P4501A1，提出了五个区域作为还原酶的假定结合位点（Shen，S.和Strobel，H. W.（1993年）《生物化学与生物物理学报》304，257 - 265）。为了阐明这些氨基酸残基各自的具体作用，针对包含8 - 13个氨基酸的这些区域产生了五种抗肽抗体，分别定义为1A、4A、5A、6A和7A，并使用肽 - 琼脂糖4B柱进行亲和纯化。通过酶联免疫吸附测定和蛋白质免疫印迹技术分析表明，五种抗肽抗体中的三种与肽以及细胞色素P4501A1具有特异性结合。将各种抗肽抗体与细胞色素P450孵育，然后与还原酶、脂质和NADPH重组，结果显示抗体5A和6A对P450活性有显著抑制作用，而1A、4A或7A则没有。抗体5A对由氢过氧化异丙苯支持的P450活性也有抑制作用，尽管在每种抗体浓度增加时，其抑制作用比还原酶支持的活性抑制作用低45%至30%。对抗体5A的动力学研究表明，底物乙氧基香豆素的Km没有变化，但无论与还原酶重组还是由氢过氧化异丙苯支持，对细胞色素P4501A1系统的Vmax都有显著影响。对抗体5A对细胞色素P4501A1影响的表征表明，抗体5A与P4501A1的结合可能会改变P4501A1与还原酶的结合。此外，5A与细胞色素P4501A1的结合也降低了P450的Vmax。这些结果与细胞色素P4501A1中从氨基酸残基269至281（肽5）和454至463（肽6）的区域在细胞色素P4501A1羟化活性中的作用一致。
