scholarly journals Effect of oxidized glutathione and temperature on inositol 1,4,5-trisphosphate binding in permeabilized hepatocytes

1995 ◽  
Vol 310 (1) ◽  
pp. 185-192 ◽  
Author(s):  
D C Renard-Rooney ◽  
S K Joseph ◽  
M B Seitz ◽  
A P Thomas
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Oxidized Glutathione ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Temperature Dependent ◽  
Inositol 1,4,5 Trisphosphate ◽  
Number Of Binding Sites

The effect of oxidized glutathione (GSSG) on inositol 1,4,5-trisphosphate (IP3) binding and the activity of IP3-gated Ca2+ channels was examined in permeabilized hepatocytes. The permeability properties of the channel were measured by using Mn2+ quenching of compartmentalized fura-2 at 37 degrees C and at 4 degrees C for comparison with IP3-binding measurements. GSSG (2 mM) increased the IP3-sensitivity of Mn2+ quenching, consistent with previous studies based on Ca(2+)-release measurements [Renard, Seitz and Thomas (1992) Biochem. J. 284, 507-512]. Measurements of [3H]IP3 binding were made at 4 degrees C after preincubation of permeabilized hepatocytes at 37 degrees C in the absence or presence of GSSG. Under these conditions GSSG stimulated IP3 binding by increasing the number of binding sites without changing the Kd. This effect was observed in the absence or presence of Ca2+, but was abolished when the preincubation with GSSG was carried out at 4 degrees C. Thimerosal also stimulated [3H]IP3 binding, but this effect was mediated both by an increase in the maximum number of binding sites and by a decrease in the Kd. The effects of thimerosal and GSSG were not additive. Further analysis of the effect of GSSG revealed that preincubation of permeabilized hepatocytes at 37 degrees C results in a progressive loss of [3H]IP3-binding sites that can be prevented and reversed by inclusion of GSSG. A parallel loss of IP3-sensitive Mn(2+)-quenchable stores was observed after incubation at 37 degrees C, and this could also be reversed by adding back GSSG. The loss of IP3 binding was not the result of IP3-receptor proteolysis, as judged by Western blotting of immunoreactive protein. The sensitivity of [3H]IP3 binding in permeabilized hepatocytes to varied ratios of GSSG and GSH suggests that the IP3 receptor responds to an oxidized redox environment such as that found in the lumen of the endoplasmic reticulum. GSSG had no direct effect on the ligand-binding activity of detergent-solubilized and partially purified IP3 receptors. We conclude that GSSG exerts an indirect effect on the IP3 receptors in permeabilized hepatocytes by preventing a temperature-dependent loss of IP3-binding sites. We suggest that the hepatic IP3 receptors may interact with a thiol-disulphide oxidoreductase that utilizes GSSG as a substrate and prevents inappropriate unfolding of the ligand-binding domain occurring after incubation of the receptor at 37 degrees C in vitro.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2645-2654 ◽  
Author(s):  
C. Yue ◽  
K.L. White ◽  
W.A. Reed ◽  
T.D. Bunch
Keyword(s):  
Ryanodine Receptors ◽  
Inhibitory Effect ◽  
Ruthenium Red ◽  
Calcium Oscillations ◽  
Ip3 Receptor ◽  
Cyclic Adp Ribose ◽  
Inositol 1,4,5 Trisphosphate ◽  
Bovine Oocytes ◽  
Continuous Injection

Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50–250 nM IP3 or 10–20 mM caffeine, 100–200 microM ryanodine and 4–8 microM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 microM. Prior injection of either 50 microM ruthenium red, 50 microM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10–20 mM caffeine or 200 microM ryanodine, but prior injection of 50 microM ruthenium red or procaine completely inhibited the effect of 10–20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 microM concentration of ruthenium red or procaine only partially blocked the effect of 200 microM ryanodine, but 200 microM ruthenium red or procaine completely blocked the effect of 200 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

THE PLATELET “REBOUND-PHENOMENON” DURING PROLONGED, CONTINUOUS PGI2 INFUSION OCCURS AT THE RECEPTOR LEVEL

1987 ◽  
Author(s):  
G Steurer ◽  
H Sinzinger ◽  
P Fitscha
Keyword(s):  
Binding Sites ◽  
Venous Blood ◽  
Intermittent Infusion ◽  
Receptor Level ◽  
Binding Data ◽  
Post Infusion ◽  
Number Of Binding Sites ◽  
Rebound Phenomenon ◽  
Infusion Regimen

During earlier attempts in optimizing the therapeutic regimen with PGI2 we were able to discover an “ intra- and post-infusion platelet rebound” being characterized by an activated platelet function and a diminished responsiveness of platelets to the action of PGI2 in-vitro.In order to verify this phenomenon at the receptor level we infused continuously 6 patients suffering from peripheral vascular disease (PVD) with PGI2 at a rate of 5 ng/kg/min for 5 days. Anticoagulated venous blood has been drawn at different intervals. Saturation binding experiments on platelet membrane fraction have been performed using [3H]iloprost, a stable PGI2 analoque. Analysis of the binding data according to Scatchard demonstrated a decrease of receptor affinity with an increased number of binding sites.It is concluded, that intrainfusion rebound occurs at the receptor level, whereas the postinfusion rebound does not. This is a further piece of evidence that an intermittent infusion regimen is preferable.

scholarly journals Interleukin-2 (IL-2) Regulates the Accessibility of the IL-2-Responsive Enhancer in the IL-2 Receptor α Gene to Transcription Factors

1999 ◽  
Vol 19 (4) ◽  
pp. 2681-2689 ◽  
Author(s):  
Corinne Rusterholz ◽  
Patricia Corthésy Henrioud ◽  
Markus Nabholz
Keyword(s):  
Transcription Factors ◽  
T Lymphocytes ◽  
Binding Sites ◽  
Interleukin 2 ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Cooperative Binding ◽  
Α Subunit ◽  
Hypersensitive Sites

ABSTRACT Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) α subunit by antigen and by IL-2 itself. IL-2 induces IL-2Rα transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2Rα expression. In cells induced to transiently express IL-2Rα with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2Rα transcription by making IL-2Rα chromatin accessible to transcription factors.

scholarly journals Agonist-dependent Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor

1999 ◽  
Vol 113 (6) ◽  
pp. 851-872 ◽  
Author(s):  
Andrew P. LeBeau ◽  
David I. Yule ◽  
Guy E. Groblewski ◽  
James Sneyd
Keyword(s):  
Acinar Cells ◽  
Calcium Oscillations ◽  
Pancreatic Acinar Cells ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Open Probability ◽  
Probability Curve ◽  
Long Period ◽  
Inositol 1,4,5 Trisphosphate ◽  
S Period

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10–12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675–2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81–84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.

Temperature Dependent Conformation Changes of Ribulose-1,5-bisphosphate Carboxylase Studied by the Use of 1-Anilino-8-naphthalene Sulfonate

1977 ◽  
Vol 32 (3-4) ◽  
pp. 226-228 ◽  
Author(s):  
Günter F. Wildner ◽  
Jürgen Henkel
Keyword(s):  
Enzyme Activity ◽  
Binding Sites ◽  
Fluorescence Probe ◽  
Fluorescence Emission ◽  
Binding Constants ◽  
Temperature Dependent ◽  
Bisphosphate Carboxylase ◽  
Influence Of Temperature On ◽  
Number Of Binding Sites ◽  
Enzyme Molecules

Abstract The influence of temperature on the structure and enzyme activity of ribulose-1,5-bisphosphate carboxylase, isolated from Euglena gracilis cells, was studied. Freezing of the purified ribulose-1,5-bisphosphate carboxylase preparation causes a severe loss of enzyme activity, which can be restored again by incubation of the enzyme molecules at higher temperatures (50 °C). The titration of both enzyme samples with the fluorescence probe 1-anilino-8-naphthalene sulfonate (ANS) re­vealed an increase of the fluorescence emission of the low temperature form of the enzyme. Two different enzyme conformations can be assumed which differ in the number of binding sites for ANS and Vmax values for the carboxylase reaction but show similar binding constants for ANS and the apparent Km values for C02 .

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites

1990 ◽  
Vol 10 (2) ◽  
pp. 859-862
Author(s):  
G M Santangelo ◽  
J Tornow
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Heterologous Gene ◽  
Dna Binding Activity ◽  
Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences

Development ◽  
1988 ◽  
Vol 104 (Supplement) ◽  
pp. 75-83 ◽  
Author(s):  
Allen Laughon ◽  
William Howell ◽  
Matthew P. Scott
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Binding Activity ◽  
Regulatory Function ◽  
Temperature Sensitive ◽  
Structural Domain ◽  
Dna Binding Activity ◽  
Dna Restriction Fragments

The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila. The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain. Here we show that the homeodomains encoded by two of the ANT-C loci confer sequencespecific DNA-binding activity. The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains. Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain. The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA. Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters. Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two. The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins

1993 ◽  
Vol 13 (4) ◽  
pp. 2354-2365
Author(s):  
K M Catron ◽  
N Iler ◽  
C Abate
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Equilibrium Constants ◽  
Binding Specificity ◽  
Binding Activity ◽  
Selection Strategy ◽  
Homeodomain Proteins ◽  
Control Regions

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

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