title: Phosphofructokinase from Dirofilaria immitis. Stimulation of activity by phosphorylation with cyclic AMP-dependent protein kinase.
abstract: Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000947:Antigens, Helminth; D001200:Ascaris / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology*; D004183:Dirofilaria immitis / Q000201:enzymology*; D000939:Epitopes / Q000276:immunology; D005370:Filarioidea / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D000947:Antigens, Helminth; D000939:Epitopes; D010710:Phosphates; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D010732:Phosphofructokinase-1