Characterization of a human placental fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase.
A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. 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 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 phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes. Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by [2-32P]fructose-2,6-bisphosphate. Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively. These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta.
Sakakibara
R
R
Department of Biochemistry, School of Pharmaceutical Sciences, Nagasaki University.
Kato
M
M
Okamura
N
N
Nakagawa
T
T
Komada
Y
Y
Tominaga
N
N
Shimojo
M
M
Fukasawa
M
M
D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010920:Placenta / Q000737:chemistry / Q000201:enzymology*; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases