[10] Phosphofructokinase from yeast

Hofmann, E; Kopperschläger, Gèrhard

doi:10.1016/s0076-6879(82)90106-9

Cited by 97 publications

(36 citation statements)

References 18 publications

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“…This value is nearly 300 × higher than in D. discoideum cells (6.5 × 10-3% [12]). The enzyme was also overexpressed with respect to native yeast PFK, since the later is ~0.5% of extract protein [33,34]. After completion of this work, we observed that the recombi- nant enzyme can also be obtained pure by omitting the polyethylene glycol fractionation step (Table 1), although with a yield of only 7%.…”

Section: Purification and Properties Of The Recombinant Enzymementioning

confidence: 90%

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

1995

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Phosphofructokinase (PFK) from yeast has been replaced by the non-allosteric isozyme from the slime mold Dictyostelium discoideum. This has been achieved by overexpression of the latter in a PFK-deficient strain of Saccharomyces cerevisiae under the control of the PFK2 promoter. Transformants complemented the glucose-negative growth phenotype exhibiting generation times on glucose-containing media similar to those of an untransformed strain being wild-type for yeast PFK genes. The PFK produced reacted with an antibody against D. discoideum PFK. It exhibited the same subunit size, quaternary structure and kinetic parameters than those of the wild-type enzyme, and was also devoid of specific regulatory properties.

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Section: Purification and Properties Of The Recombinant Enzymementioning

confidence: 90%

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

1995

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“…Phosphofructokinase was purified from a recombinate strain of Saccharomyces cerevisiae carrying a multicopy vector of the PFKI and PFK2 genes kindly supplied by Dr J. Heinisch (Dusseldorf, Germany). The enzyme was purified using the procedure described by Hofmann and Kopperschlager (1982). The concentration of solutions of purified phosphofructokinase was determined spectrophotometrically at 279 nm using a molar absorption coefficient of E = 7.36X 10'M-I .…”

Section: Reagents and Enzymesmentioning

confidence: 99%

Limited proteolysis of yeast phosphofructokinase

Kopperschläger

Bär

Stellwagen

1993

European Journal of Biochemistry

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Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteol ysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis.Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual a and p polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components.The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the a chain and Leu192 in the p chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the n chain involving the peptide bond preceding Va1914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase.It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphosphofructokinase are not directly involved in catalysis or its allosteric regulation.However, the last 80 residues of the a polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.Yeast phosphofructokinase 1 contains two kinds of polypeptide chains, an a chain containing 987 residues in a known sequence and encoded by the PFKl gene and a chain containing 959 residues, also in a known sequence encoded by the PFK2 gene (Heinisch et a]., 1989). The native enzyme is a globular protein containing four a chains and four p chains. Solution measurements indicate the native octameric enzyme has a sedimentation coefficient of 21 S and a molecular mass of about 800 kDa (Kopperschlager et al., 1977;Chaffotte et al., 1984). The octameric structure of the native enzyme is stable at a protein concentration of 1 mg/ ml at ambient temperature and neutral pH in the presence or Correspondence to

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“…Goat anti-(rabbit immunoglobulin G)-peroxidase conjugate was purchased from Nordic. Yeast phosphofructokinase was kindly donated by Dr G. Kopperschlager (University of Leipzig); it had been purified as described by Hofmann and Kopperschlager [12]. Antibodies against the M and L subunits of rat muscle and liver PFruK, respectively, and against brain PFruK were kindly donated by Dr G. A. Dunaway (Southern Illinois University).…”

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confidence: 99%

Purification and Properties of Phosphofructokinase from Dictyostelium Discoideum

Martı́nez-Costa

Estévez

Sánchez

et al. 1994

European Journal of Biochemistry

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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

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[10] Phosphofructokinase from yeast

Cited by 97 publications

References 18 publications

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

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

Limited proteolysis of yeast phosphofructokinase

Purification and Properties of Phosphofructokinase from Dictyostelium Discoideum

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