Phosphofructokinase from Dirofilaria immitis: effect of fructose 2,6-bisphosphate and AMP on the non-phosphorylated and phosphorylated forms of the enzyme.
The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis. The level of Fru-2,6-P2 was determined to be 4 +/- 0.3 nmol(g wet weight)-1 in the tissues of the filariid. Fru-2,6-P2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The Kact values for Fru-2,6-P2 were 378 +/- 18 nM and 65 +/- 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8. AMP also stimulated the activity of both forms of the enzyme with Kact values of 230 +/- 10 microM and 37.3 +/- 6.1 microM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the KFru-6-P values were 153 microM and 60 microM for the non-phosphorylated and phosphorylated forms of the PFK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Srinivasan
N G
NG
Department of Biochemistry, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Rao
G S
GS
Harris
B G
BG
D000249:Adenosine Monophosphate / Q000502:physiology*; D000818:Animals; D004183:Dirofilaria immitis / Q000201:enzymology*; D005370:Filarioidea / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism*; D006598:Hexosediphosphates / Q000378:metabolism*; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010766:Phosphorylation
D005635:Fructosediphosphates; D006598:Hexosediphosphates; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D010732:Phosphofructokinase-1