Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1.
Variations in hepatic progesterone 21-hydroxylase activity reflect differences in the microsomal concentration of rabbit cytochrome P-450 1.
复制标题
肝孕酮 21-羟化酶活性的变化反映了兔细胞色素 P-450 1 微粒体浓度的差异。
DOI：
10.1016/0003-9861(85)90253-x
复制
发表时间：
1985
影响因子：
3.9
通讯作者：
Griffin,KJ
中科院分区：
文献类型：
--
作者：
Johnson,EF;Griffin,KJ
一种特异于细胞色素P-450 - 1的单克隆抗体广泛（>95%）抑制孕酮的肝21-羟基化，用于双位点免疫放射测定，以估计从24只未处理的新西兰白色兔制备的微粒体中细胞色素P-450 - 1的浓度。这些微粒体的孕酮21-羟化酶活性范围为0.2 - 5.8 nmol min-1 mg微粒体蛋白-1。Scatchard分析揭示了相似的斜率，因此抗体和微粒体样品之间的表观亲和力在21-羟化酶活性方面变化>10倍。与低21-羟化酶活性相比，高21-羟化酶活性微粒体的抗体与不同微粒体制剂结合的最大程度更大，这表明结合水平反映了微粒体中P-450的含量1。定量是基于125 I标记的单克隆抗体与吸附在微量滴定板威尔斯孔上的异源单克隆抗体从样品中分离的P-450 1的结合程度。结果表明，P-450 - 1的含量范围为<0.05 ~ 0.5nmol/mg微粒体蛋白。该抗原的微粒体含量测定在两个网站的免疫放射分析是高度相关的（r= 0.97）与孕酮21-羟化酶活性。使用线性回归分析来估计孕酮在原位的转换数，得到的值为11 nmol脱氧皮质酮形成min-1 nmol微粒体P-450 1 - 1。这与在免疫定量试验中用作标准品的复溶纯化P-450 1在min-1 nmol-1中形成的14 nmol脱氧皮质酮值相似。
A monoclonal antibody specific for cytochromeP-450 1 that extensively (>95%) inhibits the hepatic 21-hydroxylation of progesterone was used in a two-site immunoradiometric assay to estimate the concentration of cytochromeP-450 1 in microsomes prepared from 24 individual, untreated New Zealand White rabbits. The progesterone 21-hydroxylase activities of these microsomes ranged from 0.2 to 5.8 nmol min−1mg microsomal protein−1. Scatchard analysis revealed similar slopes and thus apparent affinities between the antibody and microsome samples that varied >10-fold in 21-hydroxylase activity. The maximal extent of binding of the antibody to different microsomal preparations was greater for microsomes exhibiting high as compared to low 21-hydroxylase activity, suggesting that the level of binding reflects the microsomal content ofP-450 1. Quantitation was based on the extent of binding of the125I-labeled monoclonal antibody toP-450 1 sequestered from a sample by a heterologous monoclonal antibody adsorbed to the wells of a microtiter plate. These results indicate that the microsomal content ofP-450 1 varies from <0.05 to 0.5 nmol/mg microsomal protein. The microsomal content of this antigen as determined in the two-site immunoradiometric assay was highly correlated (r= 0.97) with progesterone 21-hydroxylase activity. Linear regression analysis was used to estimate the turnover number for progesteronein situ, yielding a value of 11 nmol deoxycorticosterone formed min−1nmol microsomalP-450 1−1. This is similar to the value of 14 nmol deoxycorticosterone formed min−1nmol−1obtained for the reconstituted, purifiedP-450 1 used as a standard in the immunoquantitation assay.