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 pM). 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.