Regulation of glycolytic enzymes in folliculogenesis

据报道，促性腺激素刺激卵泡生长和代谢。磷酸果糖激酶是糖酵解途径的关键酶，其活性在卵泡成熟过程中增加。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）虽然能催化果糖激酶最有效的变构效应物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，最近有报道称生长因子在卵泡成熟中的重要作用。在众多生长因子中，我们选择了激活素进行研究，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。以往使用的滤泡培养系统不适合测量糖酵解酶的活性变化，因为它们需要在培养基中添加血清。我们建立了体外卵泡培养系统，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-双磷酸酶活性的变化。在这个系统中，我们观察了卵泡复合物的生长和添加激活素或促性腺激素后黄体酮、雌激素和抑制素产生的变化。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。与肝脏和心脏酶相比，我们推测与卵巢酶具有最高同源性的睾丸酶缺乏camp依赖性蛋白激酶的磷酸化位点。虽然我们没有成功克隆卵巢6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶，但在大肠杆菌中表达了足够的睾丸酶并纯化到均匀性。弄清酶的三维结构对了解酶活性的调控机制至关重要。通过使用纯化的酶，我们成功地进行了6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶的结晶和x射线分析。该双功能酶的晶体结构表明，其激酶和磷酸酶活性位于不同的结构域。激酶结构域属于单核苷酸结合蛋白超家族，磷酸酶结构域属于磷酸甘油酸突变酶家族。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

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