Cloning and expression of placenta-specific PFK-2/F2,6Pase

胎盘特异性PFK-2/F2,6Pase的克隆和表达

• 批准号: 07680654
• 负责人: SAKAKIBARA Ryuzo
• 金额: $ 1.47万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680654/
• 关键词: fructose-6-phosphate, 2-kinase; fructose-2,6-bisphosphatase; fructose 2,6-bisphosphate; bifunctional enzyme; human placenta; glycolysis; expression; isozyme; 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.

果糖-6-磷酸，2-激酶/果糖-2，6-二磷酸酶（PFK-2/F2，6 ′-bisphosphatase，PFK-2/F2，6 ′-bisphosphatase）是糖酵解的关键调节酶磷酸果糖激酶的最强激活剂，催化果糖2，6-二磷酸的合成和水解。在哺乳动物组织中，如肝脏、骨骼肌、心脏和睾丸中，已经鉴定出该双功能酶的几种同工酶。胎盘作为一种器官是独特的，因为它具有肝、肠粘膜、肺、肾和内分泌腺的某些特征，并且它从胚胎组织分化和生长，在人类仅几个月的时间内达到成熟。从调控葡萄糖利用的角度阐明胎盘细胞中存在何种类型的PFK-2/F2，6 ′同工酶是值得的。本研究测定了人胎盘PFK-2/F2，6 ′-D（519个氨基酸）的全长cDNA（1756 bp）的核苷酸序列。构建表达载体，并在大肠杆菌BL 21（DE 3）中表达。表达的蛋白质，纯化到同质，显示了58，000的分子量通过凝胶电泳在变性条件下，相比，推导的分子量为59，410。N端15个氨基酸序列与推导序列一致。经分子筛过滤鉴定，活性酶为二聚体。表达的酶是双功能的，Vmax值为142和0.2毫单位/毫克的激酶和磷酸酶的活动，分别。表达的酶被cAMP依赖性蛋白激酶、蛋白激酶C和果糖-2，6-二磷酸化。在此基础上，对胎盘PFK-2/F2，6 β的功能进行了初步探讨。

项目成果

Akiko Sakai: "Cloning of cDNA encoding for a novel isozyme of fructose 6-phosphate,2-kinase/fructose 2,6-bisphosphatase from human placenta" J.Biochem.119. 506-511 (1996)

Akiko Sakai：“从人胎盘中克隆编码新型果糖 6-磷酸，2-激酶/果糖 2,6-双磷酸酶同工酶的 cDNA”J.Biochem.119。

Akiko Sakai, Mie Kato, Masashi Fukasawa, Eisuke Furuya, and Ryuzo Sakakibara: "Cloning of cDNA encoding foe a novel isozyme of fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase from human placenta" J.Biochem. 119. 506-511 (1996)

Akiko Sakai、Mie Kato、Masashi Fukasawa、Eisuke Furuya 和 Ryuzo Sakakibara：“从人胎盘中克隆编码新型果糖 6-磷酸、2-激酶/果糖 2,6-双磷酸酶同工酶的 cDNA”J.Biochem。

Yuhki Harada, Nobuaki Tominaga, Mitsuaki Watanabe, Ryoko Shimokawa, Masatsune Ishiguro, and Ryuzo Sakakibara: "Inhibition of fructose-6-phosphate, 2-kinase by N-bromo acetylethanolamine phosphate in vitro and in vivo" J.Biochem. 121, in press.

Yuhki Harada、Nobuaki Tominaga、Mitsuaki Watanabe、Ryoko Shimokawa、Masatsune Ishiguro 和 Ryuzo Sakakibara：“N-溴乙酰乙醇胺磷酸盐在体外和体内对 6-磷酸果糖 2-激酶的抑制作用”J.Biochem。

Yuhki Harada: "Inhibition of Fructose-6-phosphate,2-kinase by N-bromoacetylethanolamine phosphate in vitro and in vivo" J.Biochem.121(印刷中). (1997)

Yuhki Harada：“N-溴乙酰乙醇胺磷酸盐在体外和体内对果糖-6-磷酸，2-激酶的抑制”J.Biochem.121（出版中）。

Yuhki Harada: "Inhibition of Fructose-6 phosphate,2-kinase by N-bromoacetylethanolamine phosphate in vitro and in vivo" J.Biochem.121(in press). (1997)

Yuhki Harada：“N-溴乙酰乙醇胺磷酸盐在体外和体内对 6 磷酸果糖，2-激酶的抑制作用”J.Biochem.121（印刷中）。