Studies on the phosphorylation of muscle phosphofructokinase

Phosphorylation of rabbit skeletal muscle phosphofructokinase by the catalytic subunit of cyclic AMP-dependent protein kinase occurs with a Km of about 230 microM and Vmax approaching that seen with histone as a substrate. The rate of phosphorylation of phosphofructokinase by protein kinase is increased by allosteric activators of phosphofructokinase, whereas inhibitors of phosphofructokinase inhibit the phosphorylation. Inhibitors and activators change Vmax but not Km. The site of phosphorylation is a serine residue that is the sixth amino acid from the carboxyl terminus. Limited proteolysis by trypsin releases an octapeptide from the carboxyl terminus and a brief exposure to subtilisin releases a dodecapeptide from the carboxyl end. The sequence of the dodecapeptide is His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu-Ala-Thr-Val. Phosphofructokinase isolated from a rabbit injected 18 h prior to killing with [32P]PO4 contained covalently bound radioactive phosphate. Approximately 80% of the phosphate was released in a trichloroacetic acid-soluble form following limited proteolysis by trypsin, under which conditions the enzyme remained with a monomer size of about 80,000 daltons. The position of elution from Sephadex G-25 of the phosphopeptide was identical with that found following limited trypsin proteolysis of in vitro labeled enzyme. Migration of the phosphopeptides on thin layer cellulose chromatography was also identical. We conclude that at least 80% of the radioactive phosphate introduced within 18 h of an intravenous injection of [32P]PO4 is found at the same site as that introduced by phosphorylation with the catalytic subunit of cyclic AMP-dependent protein kinase.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Kinetic studies on the reaction catalysed by phosphofructokinase from Trypanosoma brucei.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.