Tillmann H, Eschrich K
Institute of Biochemistry, University of Leipzig, School of Medicine, Leipzig, Germany.
Gene. 1998 Jun 8;212(2):295-304. doi: 10.1016/s0378-1119(98)00181-4.
By applying a newly developed method, cDNAs for the human muscle isoform of fructose-1,6-bisphosphatase were isolated from phage- and plasmid-derived libraries. From these cDNAs and an EST clone, a composite sequence (1302 bp) was deduced that contains an open reading frame encoding a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose 2,6-bisphosphatase was found to be active in cel-free extracts and could be strongly inhibited by AMP and fructose 2,6-bisphosphate. Sequence comparisons revealed that (1) all amino acids thought to be in contact with substrate molecules, regulatory molecules or metal ions in mammalian liver fructose-1,6-bisphosphatases are, with one exception, conserved in the human muscle enzyme and (2) the human muscle isoform is more homologous to the mouse intestine fructose-1,6-bisphosphatase than to the mammalian liver isoform. This is the first report of the cloning and expression of a muscle fructose-1,6-bisphosphatase isoenzyme.
通过应用一种新开发的方法，从噬菌体和质粒衍生文库中分离出了人肌肉型果糖-1,6-二磷酸酶的cDNA。根据这些cDNA和一个EST克隆，推导得到了一个复合序列（1302 bp），该序列包含一个开放阅读框，编码一个由339个氨基酸组成的多肽，估计分子量为36755。在大肠杆菌中过表达后，发现重组人肌肉果糖2,6-二磷酸酶在无细胞提取物中有活性，并且能被AMP和果糖2,6-二磷酸强烈抑制。序列比较显示：（1）在哺乳动物肝脏果糖-1,6-二磷酸酶中，所有被认为与底物分子、调节分子或金属离子接触的氨基酸，除一个例外，在人肌肉酶中是保守的；（2）人肌肉型同工酶与小鼠肠道果糖-1,6-二磷酸酶的同源性高于与哺乳动物肝脏型同工酶的同源性。这是关于肌肉果糖-1,6-二磷酸酶同工酶克隆和表达的首次报道。
