The effects of hormones on liver fructose bisphosphatase concentration and activity: Application of a new specific radioimmunoassay (original) (raw)
Related papers
Biochimica et biophysica acta, 1972
I. Chicken muscle and chicken liver ffuctose-i,6-diphosphatases (D-fructose-1,6-diphosphate I-phosphohydrolase, EC 3.1.3.11) have been purified. The enzymes were found to be homogeneous according to several criteria including purification to a constant specific activity, electrophoresis on cellulose acetate strips, absence of other glycolytic enzyme activities, and sedimentation velocity studies. The chicken liver enzyme was also tested for homogeneity by immunodiffusion on agar and by crystallization. 2. Comparisons of the two enzymes show that they have similar ultraviolet light absorption spectra but differ with respect to several other properties. The specific activity of the pure muscle enzyme is higher than that of the pure liver enzyme. Electrophoresis in several buffers and isoelectric point determinations (liver, 8.I and muscle, 8.6) indicate that the avian enzymes differ in their electrostatic natures. The muscle enzyme (S2o,w of 7.0) sediments at a slightly faster rate than the liver enzyme (s20,w of 6.8). Immunological analysis by double diffusion on agar and quantitative precipitin tests with anti-serum to chicken liver fructose-I,6-diphosphatase indicate that the liver and breast nmscle enzymes differ immunologically. Kidney fructose-1,6-diphosphatase, in contrast, is immunologically similar to the liver enzyme. The anti-serum readily cross-reacts with other avian ffuctose-I,6-diphosphatases (turkey and budgerigar) but not with mammalian liver fructose-i,6-diphosphatases (rat and rabbit). The differences in properties of chicken liver and chicken breast muscle fructose-i,6-diphosphatases and immunological similarity of chicken liver and kidney fructose-I,6-diphosphatases suggest that there are at least two isozymic forms of avian fructose-1,6-diphosphatases.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1993
1. Kinetic parameters of human and rabbit liver D-fructose 1,6-diphosphate 1-phosphohydrolase (EC 3.1.3.11) (FDP-ase) at 25 and 37 degrees C have been determined. 2. Km determined at 25 degrees C were 1.4 microM for human and 1.6 microM for rabbit enzyme; at 37 degrees C, corresponding values were 1.7 and 1.8 microM. 3. Both enzymes are allosterically inhibited by AMP. Respective values of I0.5 were 7.2 microM for human and 13.2 microM for rabbit at 25 degrees C, and 16.6 microM for human and 27.3 microM for rabbit at 37 degrees C. 4. Fructose 2,6-diphosphate, a potent regulator of gluconeogenesis, is more effective at 25 than at 37 degrees C. Ki determined at 25 degrees C was 0.07 microM for human and 0.035 microM for rabbit in comparison with 0.17 microM for human and 0.09 microM for rabbit at 37 degrees C. 5. Affinity of FDP-ase for magnesium is also dependent on temperature. For the human enzyme, Km at 25 degrees C was 226 microM and at 37 degrees C, 176 microM. For the rabbit enzyme, corresponding values were 256 and 240 microM. 6. Both enzymes are activated by KCl. Determined values of A0.5 were 91 mM for human, and 50 mM for rabbit enzyme at 25 degrees C, and 129 mM for human and 100 mM for rabbit enzyme at 37 degrees C.
The Action of Insulin on Hepatic Metabolism * Fructose 2 , 6-Bisphosphate
2001
The inhibition of hepatocyte 6-phosphofructo-1-kinase by glucagon was suppressed by insulin when the enzyme was measured in crude extracts. However, no effect of either hormone was observed after the removal of allosteric effectors from the enzyme, suggesting that the alterations in activity may be due to changes in the level of fructose 2,6-bisphosphate, a potent allosteric activator of the enzyme. Insulin opposed the action of both glucagon and exogenous cyclic AMP to lower fructose 2,6-bisphosphate levels. The concentration of glucagon and of cyclic AMP that gave a half-maximal decrease in fructose 2,6-bisphosphate levels was increased in the presence of 10 nM insulin from 0.03 to 0.09 nM and from 12 to 36 pM, respectively. Insulin also counteracted the effect of maximal concentrations of epinephrine on fructose 2,6-bisphosphate levels. In the presence of 0.02 1 1 ~ glucagon or 10 pM epinephrine, 10 nM insulin enhanced 6-phosphofructo2-kinase and decreased fructose 2,6-bisphospha...
Changes in Effective Enzyme Concentration in the Growing Rat Liver
Journal of Biological Chemistry, 1961
As a companion study to the investigation of changes in enzyme activities during fasting and repletion (1) the present investigation is concerned with changes in the regenerating liver after removal of the greater part of that organ. A period of active hepatic anabolism is common to both situations. However, there occurs marked loss of solids, variation in enzyme composition (l), and diminution of cell volume as indicated by the constancy of deoxyribonucleic acid content (2) in the liver of the fasted animal before repletion, whereas there is normal composition of the hepatic remnant at the time of partial hepatectomy. Our previous studies emphasized the nonuniformity of regeneration of hepatic constituents after repletion after starvation (1). Other investigations (3-7) have established that not all hepatic components regenerate at the same rate after partial ablation of the liver. Thus a direct comparison of the two situations (with the use of the same methods) seemed in order, and afforded an opportunity for comparison of hepatic composition during these differing anabolic states.
Journal of Histochemistry and Cytochemistry, 1993
The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK) , glycogen synthase (GS) , and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat seaions of tissue were collected on slides, fued by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was codirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed ani-GIFFlN, DRAKE, MORRIS, CARDELL Materials and Methods Solutions. The following solutions were used: (a) PBS + TX-100: 0.137 M NaCI, 4 mM Na2HP04, 15 mM NaH2P04, 0.5% (v/v) Triton X-100, pH 7.4; (b) TBST blocking buffer: 100 mM Trizma Base, 0.9% NaCI, 0.1% (vlv) Tween-20, 5 % (wlv) Carnation non-fat dry milk, pH 7.5; (c) homogenization buffer: 20 mM Tris-HCI, pH 7.8, 150 mM potassium fluoride, 10 mM EDTA acid, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 pg/d each of leupeptin, pepstatin, and chymostatin; (d) SDS t a d buffer: 25 mM Trizma Base, pH 8.3,0.192 M glycine, 0.1% SDS; (e) electrophoretic transfer buffer: 10 mM Tris-HCI, 192 mM glycine, 10% (vlv) methanol; (f) blocking buffer: PBS + TX-100 with 2% (w/v) bovine serum albumin, 0.9% (v/v) fish gelatin, and 5% (v/v) normal rabbit serum for PEPCK and GS or normal goat serum for GP. Vectabond (Vector Laboratories; Burlingame, CA) was used to coat slides for cryostat sections. Animals. Normal adult Sprague-Dawley rats were maintained and sacrificed according to the NIH Guidelines for Care and Use of Laboratory Animals. Livers from fed or overnight fasted animals were frozen in liquid nitrogen for Western blot analysis, fixed, and processed for immunocytochemistry, or prepared for histology.
Bioscience Reports, 1986
In fetal rat liver the concentration of fructose 2,6-bisphosphate is decreased by administration of glucagon. The glucagon effect, i.e., the phosphorylation state of phosphofructokinase 2, dominates over the substrate supply. Insulin was found to increase fructose 2,6-bisphosphate only when exogenous glucose is supplied simultaneously. The total activity of phosphofructokinase 2 exhibits remarkable developmental changes. It is high at term, moderate in the fetal as well as in the mature organ, and low during suckling. The level of the enzyme during development is controlled by pancreatic and adrenal hormones.
Comparative Characterization of liver glycogen metabolism in rat and gunea-pig
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1992
Al~'aet-1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca 2+-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-I is phosphorylated by cyclic AMP-dependent protein kinase.