Does rat liver 6-phosphofructo-1-kinase exhibit 'half-of-the-sites-phosphorylation'?
6-Phosphofructo-1-kinase (PFK-1) from a variety of species and organs can undergo phosphorylation by cAMP-dependent protein kinase. In most studies the stoichiometry of the phosphorylation reaction was far below the expected minimum value of 4 mol phosphate/mol PFK-1 tetramer. The present study with rat liver PFK-1 and purified catalytic subunit of cAMP-dependent protein kinase was undertaken in order to find the maximum phosphorylation stoichiometry under well-defined conditions. Irrespective of whether PFK-1 had been first treated with purified protein phosphatase 2C or not, no more than 1.66 +/- 0.22 mol phosphate/mol PFK-1 tetramer was incorporated, the highest single value being 2 mol phosphate/PFK-1 tetramer. This stoichiometry was found to be independent from the method of protein evaluation (gel dye-binding assay or amino acid analysis) and from the concentration of PFK-1 in the phosphorylation system (15.6 nM-0.53 microM). The stoichiometry was not affected by the presence of allosteric ligands, fructose-1,6-bisphosphatase or the PFK-1-inactivating protein. The possibility could be excluded that partial proteolysis was responsible for the incomplete phosphorylation. Two-dimensional polyacrylamide gel electrophoresis gave no indication of the existence of two different subunits in rat liver PFK-1. Possible reasons why rat liver PFK-1 undergoes 'half-of-the-sites' phosphorylation are discussed.
Domenech
C E
CE
Abteilung Klinische Biochemie, Zentrum Innere Medizin Universität Göttingen, Federal Republic of Germany.
Mieskes
G
G
Söling
H D
HD
D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D004587:Electrophoresis, Agar Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases; D051381:Rats
D000596:Amino Acids; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases; D010732:Phosphofructokinase-1