Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme.
PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.
Mertens
E
E
Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Belgium.
De Jonckheere
J
J
Van Schaftingen
E
E
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D011756:Diphosphates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005635:Fructosediphosphates / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016848:Naegleria fowleri / Q000201:enzymology*; D017830:Octoxynol; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013861:Thiocyanates / Q000494:pharmacology
D003902:Detergents; D011756:Diphosphates; D005635:Fructosediphosphates; D046911:Macromolecular Substances; D013861:Thiocyanates; D011092:Polyethylene Glycols; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D017830:Octoxynol; D010732:Phosphofructokinase-1; C009941:potassium thiocyanate