Citrate and the regulation of adipose-tissue phosphofructokinase.
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ABSTRACT:
SUBMITTER: Denton RM
PROVIDER: S-EPMC1265151 | biostudies-other | 1966 Aug
REPOSITORIES: biostudies-other
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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.
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Project description:1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0.1% of the value of the ratio at pH7.4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD(+) concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.
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Project description:Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.
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