Partial purification and regulatory properties of phosphofructokinase from Aspergillus niger.
ABSTRACT: Phosphofructokinase (EC 184.108.40.206) from a citric acid-producing strain of Aspergillus niger was partially purified by the application of affinity chromatography on Blue Dextran--Sepharose and the use of fructose 6-phosphate and glycerol as stabilizers in the working buffer. The resulting preparation was still impure, but free of enzyme activities interfering with kinetic investigations. Kinetic studies showed that the enzyme exhibits high co-operativity with fructose 6-phosphate, but shows Michaelis--Menten kinetics with ATP, which inhibits at concentrations higher than those for maximal activity. Citrate and phosphoenolpyruvate inhibit the enzyme; citrate increases the substrate (fructose 6-phosphate) concentration for half-maximal velocity, [S]0.5, and the Hill coefficient, h. The inhibition by citrate is counteracted by NH4+, AMP and phosphate. Among univalent cations tested only NH4+ activates by decreasing the [S]0.5 for fructose 6-phosphate and h, but has no effect on Vmax. AMP and ADP activate at low and inhibit at high concentrations of fructose 6-phosphate, thereby decreasing the [S]0.5 for fructose 6-phosphate. Phosphate has no effect in the absence of citrate. The results indicate that phosphofructokinase from A. niger is a distinct species of this enzyme, with some properties similar to those of the yeast enzyme and in some other properties resembling the mammalian enzyme. The results of determinations of activity at substrate and effector concentrations resembling the conditions that occur in vivo support the hypothesis that the apparent insensitivity of the enzyme to citrate during the accumulation of citric acid in the fungus is due to counteraction of citrate inhibition by NH4+.
Project description:Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.
Project description:1. Phosphofructokinase from rat kidney cortex has been partially purified by using a combination of isoelectric and ammonium sulphate precipitation. This preparation was free of enzymes which interfered with the measurement of either product of phosphofructokinase. 2. At concentrations greater than the optimum, ATP caused inhibition which was decreased by raising the fructose 6-phosphate concentration. This suggested that ATP reduced the affinity of phosphofructokinase for the other substrate. Citrate potentiated the ATP inhibition. 3. AMP and fructose 1,6-diphosphate relieved the inhibition by ATP or citrate by increasing the affinity of the enzyme for fructose 6-phosphate. 4. K(+) is shown to stimulate and Ca(2+) to inhibit phosphofructokinase. 5. The similarity between the complex properties of phosphofructokinase from kidney cortex and other tissues (e.g. cardiac and skeletal muscle, brain and liver) suggests that the enzyme in kidney cortex tissue is normally subject to metabolic control, similar to that in other tissues.
Project description:1. The effects of ATP, inorganic phosphate and citrate on the relationship between fructose 6-phosphate concentration and initial velocity of reaction has been investigated with a partially purified preparation of rat-heart phosphofructokinase. 2. At low concentrations of ATP (<80mum) rate curves for fructose 6-phosphate approximated to Michaelis-Menten kinetics. At higher ATP concentrations rate curves were sigmoid, the K(m) for fructose 6-phosphate increased and the reaction appeared to be first-order with respect to fructose 6-phosphate at concentrations above its K(m) and of a higher order at concentrations below its K(m). Inorganic phosphate lowered the K(m) for fructose 6-phosphate and the concentration at which the apparent kinetic order decreased. 3. At 40mum-ATP, citrate was an activator at low concentration (<100mum) and an inhibitor at higher concentrations. At 0.5mm-ATP, citrate was inhibitory at all concentrations tested. 4. A new method for phosphofructokinase assay using [U-(14)C]fructose 6-phosphate is described which allows measurements to be made of the velocity of the forward reaction at known concentrations of the products of the reaction. With this method confirmatory evidence has been obtained that concentrations of ATP, AMP, phosphate and citrate may regulate phosphofructokinase in the perfused rat heart.
Project description:1. Ox heart phosphofructokinase catalyses isotope-exchange reactions at pH6.7 between ADP and ATP, and between fructose 6-phosphate and fructose 1,6-diphosphate, the latter reaction being absolutely dependent on the presence of the magnesium complex of ADP. 2. The reaction kinetics are hyperbolic with respect to substrate concentration for both exchange reactions (within the experimental error). 3. The influence of pH, AMP and citrate suggests that the fructose 6-phosphate-fructose 1,6-diphosphate exchange is subject to effector control, and is abolished by dissociation of the enzyme. 4. These results are discussed in relation to the reaction mechanism of the enzyme.
Project description:1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.
Project description:1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.
Project description:Phosphofructokinase (EC 220.127.116.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.
Project description:1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4-7mumoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18.5mumoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.
Project description:1. Pea-seed phosphofructokinase was purified 27-fold by a combination of fractionation with ethanol and ammonium sulphate. Under the conditions of assay, the enzyme was strongly inhibited by phosphoenolpyruvate. This inhibition was reversed by increasing the concentration of fructose 6-phosphate or magnesium chloride, or by lowering the ATP concentration. 2. Citrate, ADP and AMP inhibited phosphofructokinase and increased the sensitivity to phosphoenolpyruvate inhibition. Sulphate and inorganic phosphate stimulated the enzyme activity and decreased the sensitivity to phosphoenolpyruvate. 3. In the presence of inorganic phosphate and low concentrations of ATP, inhibition by phosphoenolpyruvate ceased and phosphoenolpyruvate became stimulatory. 4. The possible significance of these results in the control of plant carbohydrate metabolism is discussed.
Project description:A new procedure for the purification of phosphofructokinase using Blue Dextran-Sepharose is described. This allowed an approx. 1000-fold purification of phosphofructokinase from rat white and brown adipose tissue to be achieved in essentially a single step. The purified enzymes from both tissues were found to exhibit hyperbolic kinetics with fructose 6-phosphate, to be inhibited by ATP and citrate, and to be activated by 5'-AMP, phosphate and fructose 2,6-bisphosphate. The enzymes were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, and phosphorylation was found to be associated with increases in activity when the enzymes were assayed under appropriate sub-optimal conditions. In particular, the phosphorylated enzymes exhibited less inhibition by ATP and the white-adipose-tissue enzyme was more sensitive to activation by fructose 2,6-bisphosphate. It is suggested that an increase in the cytoplasmic concentration of cyclic AMP in tissues other than liver may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.