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

双功能果糖-6-磷酸，2-激酶/果糖-2，6-二磷酸酶(PFK-2/F2，6Pase)催化2，6-二磷酸果糖的合成和水解，是糖酵解的关键调节酶磷酸果糖激酶最强大的激活剂。这种双功能酶的几种同工酶已经在哺乳动物组织中被鉴定出来，如肝脏、骨骼肌、心脏和睾丸。胎盘作为一个器官是独一无二的，因为它具有肝脏、肠粘膜、肺、肾脏和内分泌腺的一些特征，它从胚胎组织分化和生长，在人类仅用几个月的时间就达到成熟。从控制葡萄糖利用的角度来看，胎盘细胞中存在什么样的PFK-2/F2，6Pase同工酶是值得阐明的。为研究人胎盘PFK-2/F2，6Pase同工酶的特性，测定了编码人胎盘PFK-2/F2，6Pase(519个氨基酸)的全长cDNA1756个核苷酸序列。构建表达载体，在大肠杆菌BL21(DE3)中进行表达。在变性条件下，凝胶电泳法显示表达蛋白的相对分子质量为58,000，而推定的相对分子质量为59,410。15个氨基酸的N端序列与推导的序列一致。经分子筛过滤鉴定，该酶为二聚体。表达的酶具有双功能，其酶活和磷酸酶活性的Vmax分别为142和0.2mU/mg。表达的酶被cAMP依赖的蛋白激酶、蛋白激酶C和2，6-二磷酸果糖所激活。在此基础上，讨论了胎盘组织中PFK-2/F2，6Pase的作用。

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.