|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
|Keywords||fructose-6-phosphate, 2-kinase / fructose-2,6-bisphosphatase / fructose 2,6-bisphosphate / bifunctional enzyme / human placenta / glycolysis / expression / isozyme / fructose 6-phosphate,2-kinase / fructose 2,6-bisphosphatase / 二機能性酵素 / ヒト胎盤 / アイソザイム / 解糖系 / 発現 / fructose 6-phoshate,2-kinase|
The bifunctional enzyme fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (PFK-2/F2,6Pase) catalyzes the synthesis and hydrolysis of fructose 2,6-bisphosphate, which is the most powerful activator of phosphofructokinase, a key regulatory enzyme of glycolysis. Several isozymes of the bifunctional enzyme have been identified in mammalian tissues, such as the liver, skeletal muscle, heart and testis. The placenta is unique as an organ in that it has some of the charasteristics of the liver, of the intestinal mucosa, of the lung, of the kidney, and the endocrine gland, and it differentiates and grows from embrionic tissue to reach maturity in a period of only months in human. It is worthwhile to elucidate what type of PFK-2/F2,6Pase isozyme exists in placental cells from the standpoint of the control of glucose utilization. The aim of this project was to characterize human placental PFK-2/F2,6Pase isozyme.
The nucleotide sequence of a full length cDNA (1756 bp), which encodes a human placental PFK-2/F2,6Pase (519 amino acids), was determined. A expression vector was constructed and expressed in Escherichia coli BL21 (DE3). The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410. The N-terminal sequence of 15 amino acids was coincided with that of the deduced sequence. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively. The expressed enzyme was phosphory lated by cAMP-dependent protein kinase, protein kinase C and also by fructose-2,6-bisphosphate. Role of the placental PFK-2/F2,6Pase was discussed based on these results obtained in this project.