Purification and Properties of Phosphofructokinase from Dictyostelium Discoideum

Abstract

Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15000-fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 95-kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells. The apparent PI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (EJ of 29

A Yeast Phosphofructokinase Insensitive to the Allosteric Activator Fructose 2,6-Bisphosphate

“…The latter enzyme is not known to be subject to any kind of allosteric regulation (46), and yet yeast transformants carrying the gene on multicopy plasmids grow like wild-type cells on media containing either glucose or gluconeogenic carbon sources (45). Finally, our data underline the importance of allosteric regulation of Pfk for yeast metabolism, a notion established previously in in vitro experiments trying to mimic the in vivo situation as closely as possible (47).…”

Functional complementation of yeast phosphofructokinase mutants by the non‐allosteric enzyme from Dictyostelium discoideum

“…PFK activity was measured by using a spectrophotometric coupled assay for the formation of fructose-l,6-P2 as described previously [12]. When PFK activity was measured during yeast growth, samples of cell culture were washed with buffer A (50 mM Hepes, 100 mM KC1, 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM NaF, 2.5/lg leupeptin, pH 6.8).…”

“…Fax: (34) (1) 3975353. tive binding sites for effectors (Est6vez, A.M., Martinez-Costa, O.H., Sfinchez, V. and Arag6n, J.J., in prep.). PFK activity in the slime mold is scarce, ~0.5-1.2 U/g wet mass [12]. Therefore, we were interested in expressing this enzyme as a heterologous protein to obtain it in high amounts, as well as to produce mutant forms by specific manipulation of the sequence, since the peculiar properties of this PFK may be of great help in understanding the structural bases of allosteric regulation.…”

“…This is a non-allosteric eukaryotic PFK that, in contrast to the enzyme from other cells, shows simple kinetic properties and lacks any regulatory mechanism other than that exerted by substrate concentration [12]. DdPFK is a homotetramer of 95-kDa subunits; it has also been cloned and sequence changes with respect to regulatory isozymes have been identified at several of the puta-*Corresponding author.…”

“…PFK from the slime mold Dictyostelium discoideum (DdPFK) has been purified recently [12]. This is a non-allosteric eukaryotic PFK that, in contrast to the enzyme from other cells, shows simple kinetic properties and lacks any regulatory mechanism other than that exerted by substrate concentration [12].…”