Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region.
Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region.
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发表时间：
1982-05
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通讯作者：
S. D. Black;Minor;Coon
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作者：
S. D. Black;Minor;Coon
已知的洗涤剂溶解的肝微粒体NADPH-细胞色素P-450还原酶保留了将电子传递给细胞色素P-450的能力，而胰酶溶解的还原酶仅将电子传递给人工受体。由于胰酶的作用失去了一个疏水片段，改变后的还原酶不再能够结合细胞色素P-450。在本研究中，对兔还原酶的主要胰酶攻击是在Lys 44-Ile 45键上，以从完整的酶(分子量为77,000)中释放出亲水结构域(分子量为71,000)。另一个片段(相对分子质量，4,800)在Lys 34-Lys 35-Lys 36序列上经历胰酶攻击，产生代表还原酶疏水结构域的多肽和作为连接区的Nona-或Decapepide(Lys 35或Lys 36-Lys 44)。该疏水性多肽来自还原酶的NH2末端，具有乙酰化的NH2末端和一个异常疏水的区域(Val 16至Phe 32)，具有预测的β-折叠结构，被认为参与了细胞色素P-450与磷脂的结合。不同的酶攻击还原酶的位置不同，但裂解点位于多肽链的一小段内。通过末端序列分析比较兔和大鼠还原酶的胰酶形式(代表亲水结构域)，显示出高度的相似性，大约80%的残基完全一致，只有Ile NH2末端附近有一个很短的可变区。
Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to transfer electrons to cytochrome P-450, whereas the trypsin-solubilized reductase transfers electrons only to artificial acceptors. Due to the loss of a hydrophobic fragment by the action of trypsin, the altered reductase is no longer capable of binding cytochrome P-450. In the present study the primary tryptic attack on the rabbit reductase was shown to be at the Lys 44-Ile 45 bond to liberate the hydrophilic domain (molecular weight, 71,000) from the intact enzyme (molecular weight, 77,000). The other fragment (molecular weight, 4,800) undergoes tryptic attack at the Lys 34-Lys 35-Lys 36 sequence to yield a polypeptide representing the hydrophobic domain of the reductase and a nona- or decapeptide (Lys 35 or Lys 36 through Lys 44) which serves as the connecting region. The hydrophobic peptide, which is derived from the NH2-terminal end of the reductase, has an acetylated NH2 terminus and a region (Val 16 through Phe 32) which is exceptionally hydrophobic, with a predicted beta-sheet structure, and is believed to be involved in the binding of cytochrome P-450 and phospholipid. The site of attack on the reductase by various proteases is different, but the cleavage points are localized within a short segment of the polypeptide chain. A comparison of the tryptic forms (representing the hydrophilic domains) of the rabbit and rat reductases by terminal sequence analysis showed a high degree of similarity, with about 80% of the residues in exact correspondence and only a short variable region near the Ile NH2 terminus.