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Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme.

ABSTRACT: 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.

SUBMITTER: Mertens E

PROVIDER: S-EPMC1134184 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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