Van Schaftingen Research Articles

Point mutations and small substitution mutations in three different upstream elements inhibit the activity of the mouse alpha 2(I) collagen promoter.

Point mutations and small substitution mutations were introduced in three different promoter segments of the mouse alpha 2(I) collagen gene that are binding sites for specific DNA-binding proteins. The first of these promoter elements contains a CCAAT motif between -80 and -84 and binds a novel factor consisting of two different components which are both needed for DNA binding (Hatamochi, A., Golumbek, P.T., Van Schaftingen, E., and de Crombrugghe, B. (1988) J. Biol. Chem. 263, 5940-5947). Four different point mutations in the CCAAT motif, which strongly inhibit binding of this heterodimeric CCAAT-binding factor, decrease the activity of the promoter at least 8-fold when assayed by DNA transfection of NIH 3T3 fibroblasts. The second cis-acting element is located around -250. Two mutations in this element, one a 3-base pair (bp), another a 4-bp substitution mutation, decrease the activity of the promoter in DNA transfection experiments by about 8- and 12-fold, respectively. The third element is located between -315 and -295 and binds nuclear factor 1 (NF1). Both 1- and 2-bp mutations which strongly inhibit binding of NF1 to this site decrease the activity of the promoter 8-10-fold in DNA transfection assays whereas another mutation in this site shows less inhibition in the activity of the promoter. Transfer of this NF1-binding site 5' to the SV40 early promoter increases the activity of this promoter more than 10-fold. These results indicate that the integrity of each of these three cis-acting elements is required for optimal activity of the alpha 2(I) collagen promoter and suggest a model whereby the factors which bind to these sequences cooperate in the formation of a transcription initiation complex.

Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: Phosphate dependence and effects of other oxyanions

The effects of various oxyanions on the activities of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) were examined. No evidence could be found for an absolute dependence of the kinase activity on inorganic phosphate as was recently reported by M. Laloux, E. Van Schaftingen, and H.-G. Hers ((1985) Eur. J. Biochem. 148, 155–159) . Rather, phosphate and arsenate activated the kinase by decreasing the enzyme's K m for fructose 6-phosphate without affecting its K m for ATP or V max. The K m of the kinase for fructose 6-phosphate in the presence of inorganic phosphate was found to be significantly lower (6 μ m) than previously reported (30 μ m) when the hydrolysis of fructose 2,6-bisphosphate by the concomitant bisphosphatase activity at low Fru 6-P concentrations was taken into account. The K A 's for phosphate and arsenate activation of the kinase were 0.2 and 0.3 m m, respectively. A number of other oxyanions, including pyrophosphate, sulfate, tungstate, selenate, and molybdate all inhibited the kinase by increasing the K m for fructose 6-phosphate. The apparent K i 's for inhibition of the kinase were in the 0.5–1 m m range. In contrast, all of these oxyanions activated the bisphosphatase, with half-maximal effects requiring millimolar concentrations. Inorganic phosphate was the most potent activator with a K A of 1 m m. In contrast to the other oxyanions, vanadate and meta-periodate inhibited the kinase but had no effect on the bisphosphatase. Vanadate appeared to be a noncompetitive inhibitor since its effects were not overcome by P i, ATP, or fructose 6-phosphate, and the species responsible was shown to be decavanadate. Like vanadate, meta-periodate had no effect on the bisphosphatase, though it was a potent inhibitor ( I 0.5 = 30 μM) of the kinase. Its effects were shown to be time-dependent and reversed by dithiothreitol, suggesting that it acted by an oxidative mechanism. These results augment the mounting body of evidence that the enzyme's two reactions are catalyzed at discrete active sites.

Effects of AMP and Fructose 2,6 Bisphosphate on Fructose 1,6 Bisphosphatase Activity in Trichoplusia ni Parasitized by the Insect Parasite, Hyposoter exiguae

The host association of the insect parasite, Hyposoter exiguae (Viereck) (Ichneumonidae), is characterized by elevated host blood sugar and fat body glycogen levels during the early period of the parasite's development and prior to direct destruction of host tissues (Thompson, 1982, Parasitology 84: 491-510). Recently, Thompson and Binder (1984, Journal of Parasitology 70: 644-651) reported that parasitized fourth instar Trichoplusia ni (Hiibner) (Noctuidae) larvae displayed a marked elevation in the activity of fat body fructose 1,6 bisphosphatase (FBPase), 1 of the rate-controlling enzymes of the gluconeogenic pathway. The results were not demonstrative but strongly suggested that the increased carbohydrate reserves may arise from an increase in gluconeogenic flux. The increased enzyme activity could not be explained on the basis of changes in the adenylate pool. Indeed, the level of adenosine 5' monophosphate (AMP), a potent inhibitor of FBPase and the major modulator of the enzyme's activity (McClard and Atkinson, 1979, Archives of Biochemistry and Biophysics 194: 236-243), was increased in parasitized individuals. The present note summarizes the results of additional experiments on the effects of AMP as well as fructose 2,6 bisphosphate (F2, 6BP) on FBPase in T. ni fat body following parasitization by H. exiguae. Fat body tissue from fourth instar T. ni larvae, 72 hr after parasitization by H. exiguae, was excised as described previously (Thompson and Binder, 1984, loc. cit.). The tissue from 10 individuals was pooled for each experiment and homogenized in 0.2 M triethanolamine buffer, pH 7.5, containing 0.5 mM EDTA and 1.0 mM MgCl2, and the homogenate centrifuged at 37,000 g for 40 min. Unparasitized larvae at the same developmental stage were used for controls. The activity of FBPase in the supernatant was determined with the enzyme-coupled assay described by Rosen, Rosen and Horecker (1965, Archives of Biochemistry and Biophysics 112: 411-420) in which the product, fructose 6 phosphate, is isomerized to the glucose derivative and oxidized in the presence of glucose 6 phosphate dehydrogenase to phosphogluconolactone accompanied by the reduction of nicotinamide adenine dinucleotide phosphate (NADP). The rate of change in optical density at 340 nm was monitored continuously with a Beckman 25 spectrophotometer. A 1 ml standard assay mixture contained: 0.02 mmoles triethanolamine, 0.5 ,moles EDTA, 1.0 umoles MgCl2, 0.5 ,umoles NADP, 1.7 units glucose 6 phosphate dehydrogenase and 3.5 units phosphoglucose isomerase. The reaction was initiated by addition of 0.2 ,umoles fructose 1,6 bisphosphate. The effects of F2,6BP concentration without and with 10 ,M AMP and AMP concentration without and with 2.5 ,uM F2,6BP were determined in fat body preparations of parasitized and control insects. The experiments were duplicated and the results expressed as a percentage of the maximal activity observed without F2,6BP and AMP, respectively (Van Schaftingen and Hers, 1981, Proceedings of the National Academy of Sciences 78: 28612863). AMP and F2,6BP were inhibitors of FBPase from T. ni fat body (Figs. 1, 2). Moreover, the inhibition caused by each effector was enhanced in the presence of the other. Although it was not possible to quantify inhibition precisely because of the variability between experiments, the effective concentration ranges of the inhibitors and the approximate levels of inhibition observed were similar to those reported for other animal systems cited below. In all cases AMP and F2,6BP were less inhibitory in fat body preparations from parasitized insects. These results were consistent with and may explain previous findings demonstrating an elevated FBPase activity in parasitized T. ni when the AMP level was simultaneously elevated. A quantitative evaluation, however, was not possible because the actual intracellular concentration of AMP was not deter-

Interference by pyruvate carboxylase in the measurement of acetyl-CoA carboxylase in crude liver preparations.

Research Article| February 15 1982 Interference by pyruvate carboxylase in the measurement of acetyl-CoA carboxylase in crude liver preparations D R Davies; D R Davies Search for other works by this author on: This Site PubMed Google Scholar E Van Schaftingen; E Van Schaftingen Search for other works by this author on: This Site PubMed Google Scholar H G Hers H G Hers Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1982) 202 (2): 559–560. https://doi.org/10.1042/bj2020559 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share MailTo Twitter LinkedIn Cite Icon Cite Get Permissions Citation D R Davies, E Van Schaftingen, H G Hers; Interference by pyruvate carboxylase in the measurement of acetyl-CoA carboxylase in crude liver preparations. Biochem J 15 February 1982; 202 (2): 559–560. doi: https://doi.org/10.1042/bj2020559 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1982 London: The Biochemical Society1982 Article PDF first page preview Close Modal You do not currently have access to this content.

Structure and configuration of fructose 2,6-bisphosphate by 31P and 13C nuclear magnetic resonance.

31P and 13C magnetic resonance spectra of fructose 2,6-bisphosphate, prepared as-described by Van Schaftingen and Hers (preceding paper)- and of commercial fructose 1,6-bisphosphate were obtained. Analysis of these spectra has confirmed the structure of fructose 2,6-bisphosphate; this ester was present as a single anomeric form to which the β configuration could be assigned.

Formation of fructose 2,6-bisphosphate from fructose 1,6-bisphosphate by intramolecular cyclisation followed by alkaline hydrolysis.

The method described by Pontis and Fischer [Pontis, H. G. and Fischer, C. L. (1963) Biochem. J. 89, 452–459] for the conversion of fructose 1-phosphate into fructose 2-phosphate has been applied to fructose 1,6-bisphosphate. This procedure allowed us to prepare fructose 2,6-bisphosphate in millimolar amounts. This ester had all the chemical and biological properties previously described for the natural stimulator of phosphofructokinase extracted from rat liver [Van Schaftingen, E., Hue, L., and Hers, H. G. (1980) Biochem. J. 192, 897–901].

Fructose 1,6-bisphosphatase in rat liver cytosol: Activation after glucagon treatment in vivo and inhibition by fructose 2,6-bisphosphate in vitro

Summary 1. After intraperitoneal injection of glucagon, the activity of fructose 1,6-bisphosphatase is increased in subsequently isolated liver cytosol from fed rats. The glucagon effect is time-dependent and is maximal 15–20 minutes after injection. At 20 minutes the activation is 44 ± 6% as compared to the controls (mean ± S.E.M., n = 16). 2. Fructose 1,6-bisphosphatase is shown to be inhibited by fructose 2,6-bisphosphate prepared according to Van Schaftingen and Hers ((1980) Biochem. Biophys. Res. Commun. 96, 1524–1531). The K i of the enzyme for fructose 2,6-bisphosphate is about 0.1 μM in control animals and is approximately doubled after injection of glucagon.

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-biphosphate.

Fructose 2,6-bisphosphate, a known powerful stimulator of phosphofructokinase [Van Schaftingen, E., Hue, L. & Hers, H.-G. (1980) Biochem. J. 192, 897-901] was found to inhibit, at micromolar concentrations, liver and muscle fructose-1,6-biphosphate (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11). The main characteristics of this inhibition are that (i) it is much stronger at low than at high substrate concentrations, (ii) it changes the substrate saturation curve from almost hyperbolic to sigmoidal, and (iii) it is synergistic with the inhibition by AMP. This inhibition may play an important role in the stimulation of gluconeogenesis by glucagon, because this hormone is known to decrease the concentration of fructose 2,6-bisphosphate in the liver [Van Schaftingen, E., Hue, L. & Hers, H.-G. (1980) Biochem. J. 192, 887-895].

Fructose 2,6-bisphosphate, the probably structure of the glucose- and glucagon-sensitive stimulator of phosphofructokinase.

The low-molecular-weight stimulator of phosphofructokinase [Van Schaftingen, Hue & Hers (1980) Biochem. J. 192, 887-895] has been purified from rat liver. It was completely destroyed upon incubation with 0.01 M-HCl for 10 min at 20 degrees C and fructose 6-phosphate and a reducing power equivalent in amount to the acid-labile organic phosphate were formed. It was therefore tentatively identified as fructose 2,6-bisphosphate.

Synthesis of a stimulator of phosphofructokinase, most likely fructose 2,6-bisphosphate, from phosphoric acid and fructose 6-phosphoric acid

A phosphoric ester having all the properties of the natural stimulator of phosphofructokinase recently isolated from rat liver and tentatively identified as fructose 2,6-bisphosphate [Van Schaftingen, E., Hue, L. & Hers, H.G. Biochem. J. 1980, in press] was formed by mixing 85% H 3PO 4 with 0.2 vol of 1.8 M-fructose 6-phosphate at 0°C or 25°C. This result indicates that no compounds other than phosphate and fructose 6-phosphate enter into the structure of the stimulator. When the reaction was carried out at 25°C, fructose 1,6-bisphosphate was also formed at a slower rate than the stimulator but for a much more prolonged period.