EXPRESSION OF HUMAN PLACENTAL AROMATASE IN SACCHAROMYCES-CEREVISIAE
EXPRESSION OF HUMAN PLACENTAL AROMATASE IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1210/mend-3-9-1477
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发表时间：
1989-09-01
影响因子：
--
通讯作者：
CHEN, S
中科院分区：
文献类型：
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作者：
POMPON, D;LIU, RYK;CHEN, S
在用芳香酶cDNA探针和寡核苷酸探针筛选人胎盘cDNA文库后，分离全长人胎盘芳香酶cDNA克隆Aro 2，所述寡核苷酸探针的序列源自人芳香酶基因组克隆。在Aro 2和两个先前描述的人芳香化酶cDNA克隆的3“-非翻译区发现了核苷酸序列的微异质性。胎盘组织中的微小序列差异和单一蛋白质种类的表达都表明芳香化酶存在不同的等位基因。用一个cDNA和两个寡核苷酸探针进行的北方印迹分析与人胎盘中由于差异加工而产生的2.9和2.5酶的两个mRNA信息一致。我们克隆了芳香化酶cDNA，构建了几个酵母表达质粒。该酶在酿酒酵母中表达。表达的活性被已知的芳香酶抑制剂4-羟基雄烯二酮抑制。通过分析来自携带质粒pHARK/VGAL的酵母的微粒体级分上的一氧化碳差谱估计2 μ g芳香酶/mg部分纯化的酵母微粒体的水平。使用[1 β]，2. β- 3 H]雄甾-4-烯-3，17-二酮作为底物时，通过用质粒pHARK/VGAL转化酵母合成的芳香酶获得了34 nM的表观Michaels-Menken常数（Km）和23 pmol [3 H]水形成/min/mg蛋白质的最大速度（Vmax）。动力学结果与人胎盘芳香化酶相似，表明酵母合成的芳香化酶将用于进一步的结构-功能研究。
A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3''-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with thetwo mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 .mu.g aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1.beta.,2.beta.-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.