| Co-Investigator(Kenkyū-buntansha) |
UYEDA Kosaku The Univ.TX Southwestern Med.Cent.at Dallas Dept.Biochem., Prof., Medical center at Dall, Professor
ABE Yumiko Gunma Univ.Sch.Med., Dept.OB/GIN., Adjunct Assist.prof., 医学部, 教務員 (70261857)
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| Budget Amount *help |
¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
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
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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
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