ISOLATION AND CHARACTERIZATION OF AN ALLELIC CDNA FOR HUMAN MUSCLE FRUCTOSE-1,6-BISPHOSPHATASE

By applying a newly developed method, cDNAs for the human muscle isofo rm of fructose-1,6-bisphosphatase were isolated from phage-and plasmid -derived libraries. From these cDNAs and an EST clone, a composite seq uence (1302 bp) was deduced that contains an open reading frame encodi ng a polypeptide of 339 amino acids with an estimated molecular weight of 36 755. After overexpression in E. coli, recombinant human muscle fructose-1,6-bisphosphatase was found to be active in cell-free extrac ts and could be strongly inhibited by AMP and fructose 2,6-bisphosphat e. Sequence comparisons revealed that (1) all amino acids thought to b e in contact with substrate molecules, regulatory molecules or metal i ons in mammalian liver fructose-1,6-bisphosphatases are, with one exce ption, conserved in the human muscle enzyme and (2) the human muscle i soform is more homologous to the mouse intestine fructose-1,6-bisphosp hatase than to the mammalian liver isoform. This is the first report o f the cloning and expression of a muscle fructose-1,6-bisphosphatase i soenzyme. (C) 1998 Elsevier Science B.V. All rights reserved.