pyrophosphate and potassium-thiocyanate

The cDNA for the PPi-dependent phosphofructo-1-kinase has been cloned and sequenced from a cDNA library prepared from the free-living amoeba Naegleria fowleri. The coding sequence of the cDNA consists of 1311 bases which translates into 437 amino acids with a molecular mass of 48095 Da. Comparison of the sequence with those of the previously described sequences of PPi-dependent phosphofructokinases from Propionibacterium freudenreichii and potato tuber revealed amino acid identities of 23 and 28% respectively and high conservation in those regions assumed to be part of the active site. The reading frame was cloned into an expression vector, which was transformed into Escherichia coli. Extracts of the transformed cells contained PPi-dependent phosphofructokinase activity that could be purified to homogeneity. The activity was lost on incubation with the chaotropic agent, KSCN, and recovered by subsequent incubation with AMP. These properties are consistent with those described by Mertens, De Jonckheere and Van Schaftingen [Biochem. J. (1993) 292, 797-803] for the enzyme prepared from Naegleria and support the idea that the cloned cDNA coded for the complete native enzyme. No nucleotide-binding motif or evidence for a nucleotide-binding site characteristic of the ATP-dependent phosphofructokinases could be found within the primary structure.

Topics: Adenosine Monophosphate; Amino Acid Sequence; Animals; Bacterial Proteins; Base Sequence; Cloning, Molecular; Codon; Diphosphates; DNA, Complementary; DNA, Protozoan; Gene Expression; Genes, Protozoan; Molecular Sequence Data; Naegleria fowleri; Open Reading Frames; Phosphofructokinase-1; Plant Proteins; Polymerase Chain Reaction; Propionibacterium; Protozoan Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Solanum tuberosum; Species Specificity; Thiocyanates

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.

Topics: Adenosine Monophosphate; Animals; Chromatography, Gel; Chromatography, Ion Exchange; Detergents; Diphosphates; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fructosediphosphates; Kinetics; Macromolecular Substances; Molecular Weight; Naegleria fowleri; Octoxynol; Phosphofructokinase-1; Polyethylene Glycols; Spectrophotometry, Ultraviolet; Substrate Specificity; Thiocyanates