PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE FROM THE AMEBA NAEGLERIA-FOWLERI, AN AMP-SENSITIVE ENZYME

PP(i)-dependent phosphofructokinase (PP(i)-PFK) was detected in extrac ts 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. PP(i)-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30-degrees-C in the presence of AMP. N. fowleri PP(i)-PFK was purified more than 1100-fold to near homogeneit y 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 sing le band, of 51 kDa. Size-exclusion chromatography revealed the existen ce of two forms: a large one (approximately 180 kDa), presumably a tet ramer, which was active, and a smaller one (approximately 45 kDa), pre sumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30-degrees-C in the pr esence of 0.5 mM AMP. Reactivation was also observed at 30-degrees-C i n the absence of AMP, particularly at higher enzyme concentration or i n the presence of poly(ethylene glycol). Inactivation of the tetrameri c enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme disp layed K(m) values of 10 and 15 muM for fructose 6-phosphate and PP(i), respectively, in the forward reaction, and of 35 and 590 muM for fruc tose 1,6-bisphosphate and P(i) in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased V(max.) about 2-f old without changing the affinity for the substrates; its half-maximal effect was observed at 2 muM.