Gehnrich S C, Gekakis N, Sul H S
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115.
J Biol Chem. 1988 Aug 25;263(24):11755-9.
Mouse liver mRNA enriched in sequences coding for liver phosphofructokinase by polysome immunoadsorption was used as a template for the synthesis of cDNA. The double-stranded cDNA was inserted into the expression vector lambda gt11 and cloned. Preliminary identification of clones containing cDNA sequences for phosphofructokinase was made by screening the library with anti-rat liver phosphofructokinase serum and horseradish peroxidase-conjugated goat anti-rabbit IgG as second antibody. Subsequently, by selecting antibodies specific to fusion proteins expressed by putative clones and by reacting with Western blots of mouse liver proteins several clones were positively identified as containing liver phosphofructokinase sequences. A cDNA clone corresponding to 2708 nucleotides of liver phosphofructokinase mRNA was further characterized and sequenced. The liver phosphofructokinase mRNA has an open reading frame of 2343 nucleotides followed by a 3'-untranslated region of 303 nucleotides. The G/C-rich (76%) portion of the 5'-untranslated region precedes a characteristic translational start site of CCGCC(AUG). The mRNA coding sequence indicates that the liver phosphofructokinase subunit is composed of 780 amino acid residues and has a Mr of 85,000. Comparison of the deduced amino acid sequence of mouse liver phosphofructokinase with the known rabbit muscle phosphofructokinase shows 68% homology. The N-half of the liver phosphofructokinase has conserved substrate binding sites for ATP and fructose-6-P. The 25 C-terminal residues, which contain the ATP inhibitory site, are the least homologous (20%) but contain a putative phosphorylation site (Arg-Arg-X-X-Ser). The liver phosphofructokinase mRNA is under nutritional and hormonal regulation. The liver phosphofructokinase mRNA level increased 4-fold when previously starved mice were refed a high carbohydrate, fat-free diet. This increase in mRNA level was blocked by 50% by the administration of dibutyryl cAMP. The induction of liver phosphofructokinase mRNA by fasting/refeeding was also diminished in streptozotocin diabetic mice.
通过多核糖体免疫吸附法富集编码肝脏磷酸果糖激酶序列的小鼠肝脏mRNA被用作合成cDNA的模板。双链cDNA被插入表达载体λgt11并进行克隆。通过用抗大鼠肝脏磷酸果糖激酶血清筛选文库以及用辣根过氧化物酶偶联的山羊抗兔IgG作为第二抗体，对含有磷酸果糖激酶cDNA序列的克隆进行初步鉴定。随后，通过选择对假定克隆表达的融合蛋白特异的抗体并与小鼠肝脏蛋白的Western印迹反应，几个克隆被明确鉴定为含有肝脏磷酸果糖激酶序列。对对应于肝脏磷酸果糖激酶mRNA 2708个核苷酸的cDNA克隆进行了进一步的特性分析和测序。肝脏磷酸果糖激酶mRNA有一个2343个核苷酸的开放阅读框，后面跟着一个303个核苷酸的3'非翻译区。5'非翻译区富含G/C（76%）的部分位于特征性的CCGCC（AUG）翻译起始位点之前。mRNA编码序列表明肝脏磷酸果糖激酶亚基由780个氨基酸残基组成，分子量为85,000。将小鼠肝脏磷酸果糖激酶推导的氨基酸序列与已知的兔肌肉磷酸果糖激酶进行比较，显示有68%的同源性。肝脏磷酸果糖激酶的N端有保守的ATP和果糖-6-磷酸底物结合位点。包含ATP抑制位点的25个C端残基同源性最低（20%），但含有一个假定的磷酸化位点（Arg-Arg-X-X-Ser）。肝脏磷酸果糖激酶mRNA受营养和激素调节。当先前饥饿的小鼠重新喂食高碳水化合物、无脂肪饮食时，肝脏磷酸果糖激酶mRNA水平增加了4倍。给予二丁酰cAMP可使mRNA水平的这种增加被阻断50%。链脲佐菌素糖尿病小鼠中禁食/再喂食对肝脏磷酸果糖激酶mRNA的诱导作用也减弱。
