人17β羟类固醇脱氢酶(17β-HSD)基因在家蚕系统中的表达(简报)

17β羟类固醇脱氢酶(17β-hydroxystero-id dehydrogenase,17β-HSD)能催化人体内类固醇性激素的形成和转化,例如它可以催化雌二醇与雌酮,雄烯二酮与睾酮之间的相互转化反应。人体内17β-HSD在胎盘组织含量最为丰富,催化17β-雌二醇等雌激素的生成,同时这些激素能够刺激乳腺瘤的增生。因此,如何抑制17β-HSD酶的过高活性,减少癌变发生的可能性,已成为目前这类癌症治疗的一个重要研究目标。目前,从胎盘组织中提取17β-HSD的方法,产量低,比活小,周期长,     

大鼠20α—羟类固醇脱氢酶在昆虫细胞中的表达

大鼠２０α羟类固醇脱氢酶（２０α－Ｈｙｄｒｏｘｙｓｔｅｒｏｉｄ ｄｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）ｃＤＮＡ片段,被插入杆状病毒的转移载体ｐＢｌｕｅＢａｃⅢ,经野生型病毒ＤＮＡ的共转染,从被转染的昆虫细胞中获得重组病毒。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析,重组病毒感染细胞有２０αＨＳＤ基因表达。感染细胞裂解液的Ｗｅｓｔｅｒｎ印迹法分析,３７ｋＤ的蛋白带被２０αＨＳＤ抗体识别。体外酶活性测定发现,     

大鼠20α羟类固醇脱氢酶在大肠杆菌中的表达

利用谷胱甘肽Ｓ－转移酶融合基因表达系统,在鼠２０α羟类固醇脱氢酶在大肠杆菌中得以成功地表达,亲和层析和Ｔｈｒｏｍｂｉｎ消化,可从融合蛋白中回收和纯化重组２０αＨＳＤ。ＳＤＳ－ＰＡＧＥ,Ｗｅｓｔｅｒｎ印迹法和酶活性测定显示,重组２０α ＨＳＤ具有天然蛋白质相同分子量、相似的抗原性和酶催化活性,其中ＮＡＤＰ的Ｋｍ和Ｖｍａｘ分别为９．５μｍｏｌ／Ｌ、３３４ｎｍｏｌ／（ｍｉｎ．ｍｇ）,对底物２０α羟孕酮的     

大鼠20 α－羟类固醇脱氢酶在昆虫细胞中的表达

大鼠２０α羟类固醇脱氢酶（２０α－Ｈｙｄｒｏｘｙｓｔｅｒｏｉｄｄｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）ｃＤＮＡ片段,被插入杆状病毒（ＢａｃｕＩｏｖｉｒｕｓ）的转移载体ｐＢｌｕｅＢａｃⅢ,经野生型病毒ＤＮＡ的共转染,从被转染的昆虫细胞中获得重组病毒。Ｎｏｒｔｈｅｒｎｂｌｏｔ分析,重组病毒感染细胞有２０αＨＳＤ基因表达。感染细胞裂解液的Ｗｅｓｔｅｒｎ印迹法分析,３７ｋＤ的蛋白带被２０αＨＳＤ抗体识别．体外酶活性测定发现,感染细胞裂解液中含有２０αＨＳＤ酶促活性以上结果提示,大鼠２０αＨＳＤ在杆状病毒昆虫表达系统成功地获得表达,为今后大量制备和纯化２０αＨＳＤ创造条件。     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由Ｈ ＳＤ１７Ｂ１基因编码的人Ⅰ型１７β－羟类固醇脱氢酶（１７β－ｈｙｄｒｏｘｙｓｔｅｒｏｉｄｄｅｈｙｄｒｏｇｅｎａｓｅｔｙｐｅ１简称Ⅰ型１７ＨＳＤ）催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简化（ｃＡＭＰ）对该酶在培养的绒癌胞系（ＪＡＲ和ＪＥＧ－３）中表达的调节作用。用８－ｂｒｏｍｏ－ｃＡＭＰ处理两种绒癌细胞后,观察到在伴随１．３ｋｂⅠ型１７ＨＳＤｍＲＮＡ表达的同时,Ｉ型１７ＨＳＤ蛋白浓度     

环腺苷一磷酸对人Ⅰ型17β羟类固醇脱氢酶在绒癌细胞系中表达的调节作用

由HSD17B1基因编码的人Ⅰ型17β-羟类固醇脱氢酶(17β-hydroxysteroid dehydrogenasetype 1,简称Ⅰ型17HSD)催化雌酮与雌二醇之间的转化。本文研究环腺苷一磷酸简称(cAM-P)对该酶在培养的绒癌细胞系(JAR和JEG-3)中表达的调节作用。用8-bromo-cAMP处理两种绒癌细胞后,观察到在伴随1.3 kbⅠ型17 HSDmRNA表达的同时,Ⅰ型17 HSD蛋白浓度也显著上升。标记基因分析表明,cAMP可诱导HSD 17 B1基因启动子在JAR和JEG-3细胞系中的转录活性,参与调节这一诱导作用的区域位于HSD 17 B1基因编码区上游-659至-550处。凝胶阻滞实验显示这一区域可同JAR、JEG-3、T-47 D和HeLa细胞核抽提物形成特异的DNA-蛋白复合物。本结果首次证实cAMP激活HSD 17 B1基因启动子在绒癌细胞中的转录。     

17β-羟基类固醇脱氢酶

17β－羟基类固醇脱氢酶（17β HSD）为性激素合成中最后步骤的酶,催化氧化或还原反应,其作用是在受体前调节性激素的局部水平。各型17β HSD在体内有其特有的区域性分布,分布的特点与各自的功能有着密切的关系。17β HSD结构和功能的异常与一些疾病,如肿瘤、假两性等的发生有密切关系。     

睾酮假单胞菌3α-羟类固醇脱氢酶基因的质粒载体构建及表达

从土壤中分离睾酮假单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶（3α-hsd）基因,将扩增产物用NdeⅠ/BamHⅠ消化,切下目的基因片段克隆到质粒pET-15b中构建重组pET-15b.将重组pET-15b转化入E.coli DH5α中,经酶谱分析和测序,鉴定出正确的重组质粒pET-15b.将重组pET-15b转化入宿主菌E.coli BL21(DE3) pLysS中,用硫代半乳糖苷(IPTG)进行诱导表达.提取细菌总蛋白质进行SDS-聚丙烯酰胺凝胶电泳(PAGE)分析并测定酶活性.粗提物中酶活性高达2.45×10⁵ U/L.利用重组蛋白中的6个组氨酸(His)组成的“标签”进行亲和层析,经一步金属螯合亲和层析纯化后,重组蛋白在SDS-PAGE上呈现出均一的单一条带,回收率达68%.活性和纯度均较高的目的蛋白3α-HSD的获得,为血清总胆汁酸酶循环法测定奠定了基础.     

11β—羟基类固醇脱氢酶

现已发现两型１１β－羟基因固醇脱氢酶（１１β－ＨＳＤ）：１１β－ＨＳＤ１为氧化还原酶,催化皮质醇与其代谢产物之间的相互转化;１１β－ＨＳＤ２则为专一氧化酶,只催化皮质醇的失活。１１β－ＨＳＤ１在体内分布广泛,功能目前沿不清楚。１１β－ＨＳＤ２主要存在于盐皮质激素靶器官,肾脏１１β－ＨＳＤ２通过降解糖皮质激素保护盐皮质激素受体的特异性,肾脏此酶的缺乏,可以导致严重高血压。胎盘１１β－ＨＳＤ２通过降解     

3α-羟类固醇脱氢酶的表达、纯化和酶学性质研究

从土壤中分离睾酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶（3α-HSD）基因。以质粒pET15b为载体,构建3α-HSD的原核表达系统。经IPTG诱导后,得到了具有酶活性的融合蛋白,细菌总蛋白的表达量为0.73g/L,其中融合酶蛋白占16.4%,活性达1.38×10.5U/L。利用融合蛋白N末端的Histag,经金属螯合亲和层析纯化后,得到了纯度较高的融合酶蛋白,酶蛋白的得率为68%,比活性为194.7U/mg。凝血酶切除Histag后,经质谱鉴定,重组蛋白的分子量为26.5kD,与理论推测值基本相同。25℃以雄酮为底物,以NAD+和thioNAD+为辅因子时,融合蛋白参与酶促反应的最适pH分别为10.5和9.4。37℃条件下酶促反应较快,25℃时的反应速度只有37℃时的52％,Q10（25℃～35℃）为1.7。25℃、pH105、以雄酮和各级胆汁酸为底物、NAD+为辅因子时,融合酶蛋白的动力学常数Km位于4.2～51.1μmol/L范围内。融合酶蛋白发挥催化作用并不需要金属离子的参与,而Fe3+、Fe2+、Zn2+则抑制酶活性,反应体系中加入EDTA也并不影响酶的活性。活性和纯度均较高的融合蛋白3α-HSD的获得及其生物学性质的研究,为血清总胆汁酸酶循环法测定的建立奠定了基础。     

Immunological measurement of human 17 beta-hydroxysteroid dehydrogenase

Human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) was purified to apparent homogeneity using ammonium sulfate precipitation and chromatography on Red-Agarose and DEAE-Sepharose columns. Electrophoresis on polyacrylamide gels under denaturing conditions and using silver staining showed a single protein with an apparent molecular weight of 37,800. Antibodies to the purified protein were raised in rabbits and were found by immunoblotting to be specific to 17-HSD. A sensitive radioimmunoassay was established using 125I-labeled 17-HSD as a tracer, an appropriate dilution of the antibody, and a kaolin-coupled double antibody for separating the antibody-bound and free fractions. The detection limit of the assay was approximately 150 pg/tube (1.5 micrograms/l). The cytosol fraction (105,000 g) of term placental tissue contained approximately 0.7 mg of 17-HSD per gram of protein, and the concentrations of 17-HSD measured by immunoassay and enzymatic activity proved to be strictly parallel in different partly purified placental preparations. The supernatants from centrifugations of human endometrial homogenates at 800 g and 105,000 g (after detergent treatment) displayed cross-reactivity with the antibody. The mean concentration of the cross-reacting substance in the radioimmunoassay was 14.1 micrograms/g protein (range 2-62.3) in specimens taken on different days in the cycle. These concentrations showed a significant correlation with the 17-HSD activities measured in the endometrial specimens (r = 0.722, P less than 0.001, n = 21). Mean concentrations of substance were 8.3 micrograms/g protein in endometrial specimens taken during the follicular phase (days 4-12, n = 8) and 22.9 micrograms/g protein during the luteal phase (days 16-22, n = 6) were obtained using the radioimmunoassay. There was excellent parallelism between the competition curves for [125I]iodo-17-HSD with purified 17-HSD standards and placental and endometrial homogenate dilutions. These data strongly suggest that the substance measured in the endometrial specimens was 17-HSD.     

Localization of 17 beta-hydroxysteroid dehydrogenase throughout gestation in human placenta

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) is the enzyme responsible for the formation of all sex steroids in gonadal as well as extragonadal tissues. To obtain more information about the age-specific expression of 17 beta-HSD in the human placenta, we have localized this enzyme by immunocytochemistry at the light microscopic level at different periods of gestation. In the 7- and 9-week-old placenta, immunostaining was detected exclusively in the cytoplasm of the syncytiotrophoblast. Between the tenth and thirteenth weeks of gestation, immunolabeling was also observed in the cytoplasm of the cytotrophoblastic cells, suggesting that these cells could be transiently involved in the biosynthesis of sex steroids. Interestingly, between the fourteenth and twenty-fifth weeks of gestation, 17 beta-HSD was observed in both the cytoplasm and nucleus of the syncytiotrophoblast. The reaction product was much more intense in nuclei than in cytoplasm. During the last trimester of gestation, strong immunocytochemical staining was observed in all the nuclei of the syncytiotrophoblast, the cytoplasm being unstained. The meaning of this nuclear staining for 17 beta-HSD is still unclear and remains to be extensively investigated.     

Structure of two in tandem human 17 beta-hydroxysteroid dehydrogenase genes

Two human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) genes (h17 beta-HSDI and h17 beta-HSDII) included in tandem within an approximately 13 kilobase pair fragment were isolated from a genomic lambda EMBL3 DNA library using cDNA encoding human 17 beta-HSD (hpE2DH216) as probe. We have determined the complete exon and intron sequences of the two genes as well as their 5' and 3'-flanking regions. Human 17 beta-HSDII contains six exons and five short introns for a total length of 3250 base pairs. The exon sequence of h17 beta-HSDII is identical to the previously reported hpE2DH216 cDNA while the overlapping nucleotide sequences of the corresponding exons and introns of h17 beta-HSDI and h17 beta-HSDII show 89% homology. In addition, we have used the hpE2DH216 cDNA to demonstrate the widespread expression of 17 beta-HSD mRNAs in steroidogenic and peripheral target tissues. These new findings provide the basis for a better understanding of the molecular mechanisms involved in 17 beta-HSD deficiency and peripheral sex steroid metabolism.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

Activation of 17beta-hydroxysteroid dehydrogenase from human red blood cells

Experiments designed to elucidate the nature of 17β-hydroxysteroid dehydrogenase from human red blood cells have shown that NADP⁺ activates and protects the enzyme, while also serving as substrate for the reaction. Enzyme activity was measured by the conversion of 17β-estradiol to estrone and by the production of NADPH with 17β-estradiol-3-sulfate as substrate. It appears that the reaction sequence is first, binding with NADP⁺ and second, binding with the steroid. The binding with NADP⁺ is essentially irreversible: the activated enzyme is completely protected against loss of activity by dilution. On dilution of the unactivated enzyme, much of the activity is lost. The bireactant rate equation of the sequential type has been restated for the case of activation by one of the reactants. Since it has been found that activation of enzyme is linear with NADP⁺ concentration, it follows that the Michaelis constant for the steroid substrate is independent of the concentration of NADP⁺ activating the enzyme. This is substantiated by the determination of the Michaelis constant for 17β-estradiol-3-sulfate from data on double-reciprocal plots of activated and unactivated enzyme with limiting amounts of steroid. The activating effect increases linearly up to a concentration of 1.2 × 10^?5m of NADP⁺ and then levels off. The activation is highly specific for NADP⁺; neither NAD⁺, ATP, NADPH, nicotinic acid, ncr nicotinamide prevent the loss of activity after storing the enzyme for 1 hr at 37 °C. The steroid substrate appears to interfere with the activation of NADP⁺.     

17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively.