Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from beta-naphthoflavone-fed rainbow trout.
Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from beta-naphthoflavone-fed rainbow trout.
复制标题
由来自β-萘黄酮喂养的虹鳟鱼的细胞色素 P-450 的纯化同工酶催化的苯并[a]芘羟化酶。
DOI：
10.1016/0006-2952(84)90035-2
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发表时间：
1984
影响因子：
5.8
通讯作者：
Buhler,DR
中科院分区：
文献类型：
--
作者：
Williams,DE;Buhler,DR
我们从喂食 β-萘黄酮的虹鳟鱼中纯化了五种肝微粒体 (LM) P-450 同工酶。四种形式（LM3、LM1、LM4a 和 LMx）在 DEAE-Sepharose 上分离。羟基磷灰石色谱进一步将 LMxin 分解为两种组分：LM2 和 LM4b。后一种形式以最高收率 (5%) 获得，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS-PAGE) 测定，表观最小分子量 (Mr) 为 58,000，比含量为 11.9 nmoles/mg，一氧化碳连接的 λmax，减少的差异光谱为 447.0 nm，并且在重构系统中对苯并[a]芘具有活性。第二种形式 LM4a，最终产率为 2%，比含量为 10.3，与 Lm4bbyMr、λmax 或苯并[a]芘活性无法区分。 LM2型(2％收率)的比含量为10.8，aMrof为54,000，λmax为449.5nm，并且在苯并[a]芘羟化酶的重构中无效。此外，还获得了另外两种比含量较低的形式，lm1和LM3。 lm1 和 LM3 对苯并[a]芘均没有活性。借助从兔子制备的抗体，进一步检查了 LM2、LM4a 和 LM4b 的特性。 LM4a 和 LM4b 的抗体各自与另一种抗原发生交叉反应，并在 Ouchterlony 平板上形成同一性线，并且两种 IgG 均表现出与大鼠 P-448 的一些交叉反应。两种抗体均不与鳟鱼 LM2 发生交叉反应，并且 LM2-IgG 也不与任何其他纯化的 P-450 发生交叉反应。由 LM4a 或 LM4b 催化的苯并[a]芘羟化酶被 LM4b-IgG 抑制，但不被 LM4a-IgG 抑制，表明这些抗体识别不同的抗原位点。通过氨基酸组成、肽图谱、动力学性质、对 α-萘黄酮的敏感性以及对苯并[a]-芘-二氢二醇形成的区域选择性进一步比较 LM4a 和 LM4b，表明这些形式在结构和功能上高度相似。
We have purified five isozymes of liver microsomal (LM) P-450 from β-naphthoflavonefed rainbow trout. Four forms (LM3, LM1, LM4aand LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMxinto two components, LM2and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a λmaxin the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4bbyMr, λmax, or activity towards benzo[a]pyrene. Form LM2(2% yield) had a specific content of 10.8, aMrof 54,000, a λmaxof 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, lm1and LM3. Neither lm1nor LM3was active towards benzo[a]pyrene. The properties of LM2, LM4aand LM4b, were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4aand LM4b, each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4aor LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4aand LM4bby amino acid composition, peptide mapping, kinetic properties, sensitivity to α-naphthoflavone, and regioselectivity towards benzo[a]-pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.