Regulation of glycolytic enzymes in folliculogenesis

卵泡发生中糖酵解酶的调节

• 批准号: 08044244
• 负责人: IBUKI Yoshito
• 金额: $ 0.7万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044244/
• 关键词: follicular growth; glycolysis; 6-phosphofructo-2-kinase; fructose-2,6-bisphosphatase; activin; 卵胞発育; 6-phosphofructo-2kinase

It has been reported that gonadotropins stimulate follicular growth and metabolism. The activity of phosphofructokinase, which is the key enzyme of the glycolytic pathway, increases during follicular maturation. Although 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (EC2.7.1.1.05/EC3.1.3.46) catalyzes Fru-2,6-P _2, which is the most potent allosteric effector of phosphofructokinase, there is little known of the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in the ovary. Recently however the important role of growth factors in follicular maturation has been reported. Among the many growth factors, we chose to study activin, because activin is produced in follicular granulosa cells, is the potent modulator of follicular maturation, and is involved in glucose metabolism. The follicular culture systems previously used were not suitable for measuring the change of activity of glycolytic enzymes because they require the addition of serum in the culture medium. We establ … More ished the in vitro follicular culture system with a defined medium which is useful in the monitoring of both follicular maturation and change of activity of 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase. In this system we observed the growth of the oocyte-follicular complex and the change of production of progesterone, estrogen and inhibin by the addition of activin or gonadotropins. The change of the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is an on going study. Different tissues contain tissue-specific isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The activity of liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is regulated by phospharylation. In contrast to liver and heart enzymes, the testis enzyme, which we speculate has the highest homology with the ovarian enzyme, lacks a phosphorylation site for cAMP-dependent protein kinase. Although we were not successful in the cloning of ovarian 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, enough of the testis enzyme was expressed in E coli and purified to homogeneity. It is of upmost importance to clarify the three dimensional structure of the enzyme in order to understand the regulatory mechanisms of the enzyme's activity. By using the purified enzyme, we were successful in the crystallization and X-ray analysis of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The crystal structure of this bifunctional enzyme revealed that the kinase and phosphatase activities are found in separate domains. The kinase domain was related to the superfamily of mononucleotide binding proteins and the phosphatase domain was member of the phosphoglycerate mutase family. This structure will lead to the design of specific experiments to test the mechanisms of catalysis, and ultimately, may lead to the design of innovative therapeutic compounds to alter the rate of glycolysis in the ovary. Less

据报道，促性腺激素刺激卵泡生长和代谢。糖酵解途径中的关键酶磷酸果糖激酶活性在卵泡成熟过程中升高。尽管6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶（EC2.7.1.1.05/EC3.1.3.46）催化磷酸果糖激酶最强的变构效应物Fru-2，6-P_2，但对6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶在卵巢中的作用知之甚少。然而，最近的重要作用的生长因子在卵泡成熟已被报道。在众多的生长因子中，我们选择研究激活素，因为激活素产生于卵泡颗粒细胞，是卵泡成熟的有效调节剂，并参与葡萄糖代谢。以前使用的卵泡培养系统不适合测量糖酵解酶活性的变化，因为它们需要在培养基中加入血清。我们建立了 ...更多信息 建立了卵泡体外培养系统，该系统可用于监测卵泡成熟和6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶活性的变化。在该系统中，我们观察了卵母细胞-卵泡复合体的生长以及添加激活素或促性腺激素后孕酮、雌激素和雌二醇的变化。6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶活性的变化是一个正在进行的研究。不同组织含有6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶的组织特异性同工酶。肝脏和心脏6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶的活性受磷酸化调节。与肝脏和心脏酶相反，睾丸酶，我们推测与卵巢酶具有最高的同源性，缺乏cAMP依赖性蛋白激酶的磷酸化位点。虽然我们没有成功地克隆卵巢6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶，足够的睾丸酶在大肠杆菌中表达和纯化到同质性。阐明酶的三维结构对于理解酶活性的调控机制至关重要。利用纯化的酶，我们成功地在6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶的结晶和X-射线分析。这种双功能酶的晶体结构揭示了激酶和磷酸酶活性在不同的结构域中被发现。激酶结构域与单核苷酸肽结合蛋白超家族相关，磷酸酶结构域是磷酸甘油酸酯结合蛋白家族的成员。这种结构将导致设计特定的实验来测试催化机制，并最终可能导致设计创新的治疗化合物来改变卵巢中糖酵解的速率。少

项目成果

charles A Hasemann: "The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies" structure. 4(9). 1017-1029 (1996)

查尔斯·A·哈斯曼（charles A Hasemann）：“双功能酶 6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶的晶体结构揭示了不同的结构域同源性”结构。

Y.Hasegawa: "Inhibin and Activin : Novel Requlators for Gonadal Function and Sexual Maturation" Ares-Serono Symposia Publications, 16 (1996)

Y.Hasekawa：“抑制素和激活素：性腺功能和性成熟的新型调节剂”Ares-Serono Symposia Publications，16 (1996)

T.Minegishi: "Requlation of Follicle-Stimilating Hormone Receptor" Hormone Research. 46 (suppl 1). 37-44 (1996)

T.Minegishi：“卵泡刺激激素受体的调节”激素研究。

T.Minegishi: "Requlation of Midkine Messenger Ribonucleic Acid Levels in Cultured Rat Granuloca Cells" Biochem.Biophys.Res.Comm.229. 799-805 (1996)

T.Minegishi：“培养的大鼠颗粒细胞中中期因子信使核糖核酸水平的调节”Biochem.Biophys.Res.Comm.229。

Y.Hasegawa: "Isolation and characterization of inhibin and inhibin-related proteins." J. Reprod. Develop.42(Suppl.). 23-25 (1996)

Y.Hasekawa：“抑制素和抑制素相关蛋白的分离和表征。”