Chakraborty C, Davis D L, Dey S K
Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City.
J Steroid Biochem. 1990 Feb;35(2):209-18. doi: 10.1016/0022-4731(90)90277-y.
Pig blastocysts have the capacity to convert estradiol into catechol estrogens. Our present study shows that they also have the capacity to hydroxylate estradiol in cycloaliphatic C-atom 15, and this aliphatic hydroxylation reaction is more predominate than the aromatic hydroxylations. The conversion of [4-14C]estradiol to [4-14C]15 alpha-hydroxyestradiol by mitochondrial-rich/microsomal fractions was examined by isolation of this product using reversed phase high-performance liquid chromatography (HPLC) attached to a radiometric flow detector, and its identification by gas chromatography-mass spectrometry. The enzyme kinetics for estrogen 15 alpha-hydroxylase were performed in the pig blastocyst obtained on Day 13 of pregnancy (Day 0 = first acceptance of the male). The enzyme follows classical Michaelis-Menten kinetics. The apparent Kms for estradiol were 2.47 and 1.85 microM, and the apparent Vmaxs were 0.25 and 0.197 nmol/mg/min in the mitochondrial-rich and microsomal fractions, respectively. The enzyme activity was inhibited by different steroidal compounds and non-steroidal estrogens, as well as by CO, SKF-525A, piperonyl butoxide and antibody to cytochrome P450 reductase. Ontogenesis of the blastocyst's estrogen 15 alpha-hydroxylase follows a similar pattern to that of estrogen-2/4-hydroxylase. Thus, highest activity was observed on Days 12 and 13 and lowest was on Day 15 of pregnancy. Furthermore, the enzyme is abundant primarily in the extraembryonic tissues rather than in the embryo proper. The abundance of the enzyme in the extraembryonic tissues, and its surge at a critical time of pregnancy recognition and just prior to implantation suggest that 15 alpha-hydroxylated estradiol could be involved in these processes.
猪囊胚具有将雌二醇转化为儿茶酚雌激素的能力。我们目前的研究表明，它们还具有将雌二醇的脂环族碳原子15进行羟基化的能力，并且这种脂肪族羟基化反应比芳香族羟基化反应更为显著。通过使用连接放射性流量检测器的反相高效液相色谱法（HPLC）分离产物，并通过气相色谱 - 质谱法进行鉴定，研究了富含线粒体/微粒体部分将[4-14C]雌二醇转化为[4-14C]15α-羟基雌二醇的过程。在妊娠第13天（第0天=首次接受雄性）获得的猪囊胚中进行了雌激素15α-羟化酶的酶动力学研究。该酶遵循经典的米氏动力学。在富含线粒体和微粒体部分中，雌二醇的表观Km分别为2.47和1.85微摩尔，表观Vmax分别为0.25和0.197纳摩尔/毫克/分钟。该酶活性受到不同甾体化合物、非甾体雌激素以及一氧化碳、SKF-525A、胡椒基丁醚和细胞色素P450还原酶抗体的抑制。囊胚雌激素15α-羟化酶的个体发生模式与雌激素-2/4-羟化酶相似。因此，在妊娠第12天和第13天观察到最高活性，而在妊娠第15天最低。此外，该酶主要在胚外组织中丰富，而不是在胚胎本身中。该酶在胚外组织中的丰富程度及其在妊娠识别关键时期和植入前的激增表明，15α-羟基化雌二醇可能参与这些过程。
