Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase.
Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.
Kitamura
K
K
Research Service, Veterans Administration Medical Center, Dallas, Texas 75216.
Uyeda
K
K
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D010770:Phosphotransferases; D011494:Protein Kinases; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin