CHARACTERIZATION OF A HUMAN PLACENTAL FRUCTOSE-6-PHOSPHATE 2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE

A full-length cDNA, which encodes a human placental -6-phosphate,2-kin ase/fructose-2,6-bisphosphatase, was constructed and expressed in Esch erichia coli, The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing co nditions, compared to the deduced molecular weight of 59,410. The N-te rminal sequence of 15 amino acids coincided with that of the deduced s equence, The active enzyme was a dimer as judged by molecular sieve fi ltration, The expressed enzyme was bifunctional with V-max values of 1 42 and 0.2 milliunits/mg for the kinase and phosphatase activities, re spectively. The phosphatase activity was extremely low, because one ph osphatase active site residue was mutated, and consequently the kinase /phosphatase ratio was the highest among the known isozymes, Furthermo re, the enzyme was phosphorylated by cAMP-dependent protein kinase, pr otein kinase C and also by [2-P-32] fructose-2,6-bisphosphate. Phospho rylation by cAMP-dependent protein kinase and protein kinase C increas ed the maximal Fru-6-P,a-kinase activities by 1.8- and 1.1-fold, respe ctively. These results suggested that placental e-6-phosphate,2-kinase /fructose-2,6-bisphosphatase is important in maintaining and regulatin g a relatively high rate of glycolysis in placenta.