scholarly journals Identification of a Nucleic Acid-Regulated Cyclic GMP-Binding Activity in Dictyostelium Discoideum

1989 ◽  
Vol 92 (2) ◽  
pp. 291-301
Author(s):  
A. M. PARISSENTI ◽  
M. B. COUKELL
Keyword(s):  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Rnase A ◽  
Appreciable Effect ◽  
Cell Extracts ◽  
Type Activity ◽  
Kinetics Of

Using ion-exchange chromatography, we have identified and isolated two forms of a cyclic GMP-specific binding activity in filter-broken cell extracts of Dictyostelium discoideum. Upon addition of excess cold ligand, one form (S-type) released bound 3H-labelled cyclic GMP very slowly (t½ ≈ 68 min), while the other form (F-type) released the cyclic GMP in <1 min. After photoaffinity labelling with 32P-labelled cyclic GMP, both forms revealed a major 160x103Mr band (and a few bands of lower molecular weight) on autoradiograms of sodium dodecyl sulphate-polyacrylamide gels. Addition of 500 mM-NaCl to S-type activity converted the activity to a fast-dissociating form indistinguishable from F-type, and this conversion was reversed by dialysis. Salt treatment or dialysis had no appreciable effect on the association/dissociation kinetics of F-type activity. When crude S-type activity was heated (to destroy cyclic GMP binding) and then added to F-type activity, the latter activity acquired slow-dissociating properties identical to S-type. This result suggested that the cells possess a ‘factor’ that can dramatically alter binding properties of this cyclic GMP-binding protein. Crude preparations of this factor were by boiling or proteases, but were sensitive to RNase A. Further studies revealed that acids (in particular, DNA) could effectively mimic the factor in its ability to modulate the binding kinetics of the cyclic GMP-binding activity.

Positive and negative regulation of basal expression of a yeast HSP70 gene

1989 ◽  
Vol 9 (5) ◽  
pp. 2025-2033
Author(s):  
H O Park ◽  
E A Craig
Keyword(s):  
Heat Shock ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Binding Activity ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Basal Expression ◽  
Cell Extracts ◽  
Dna Binding Assay ◽  
Kinetics Of

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.

scholarly journals Nuclear polyadenylate-binding protein.

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996 ◽  
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

Nuclear polyadenylate-binding protein

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

scholarly journals Characterization of colchicine-binding activity in Dictyostelium discoideum

1978 ◽  
Vol 169 (3) ◽  
pp. 499-504 ◽  
Author(s):  
P Cappuccinelli ◽  
B D Hames
Keyword(s):  
Dictyostelium Discoideum ◽  
High Speed ◽  
Binding Protein ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Saturation Kinetics ◽  
Filter Assay ◽  
Kinetics Of ◽  
High Speed Supernatant

A colchicine-binding component was detected in vegetative amoebae of Dictyostelium discoideum by using a Millipore-filter assay. The colchicine-binding activity is temperature-and time-dependent, maximum binding occurring at 22-35 degrees C after 60 min incubation. Further increases in temperature are without effect on the extent of binding, but bound colchicine is released with increased time of incubation. Furthermore, colchicine-binding activity itself decreased in the high-speed supernatant from D. discoideum, with half the activity being lost in approx. 2.5h. Several lines of evidence, including the saturation kinetics of colchicine binding, enhancement of colchicine binding by tartrate, insensitivity to lumicolchicine, precipitation of the binding protein by vinblastine and behaviour of the binding protein on DEAE-cellulose and Sephadex resins, suggest that the colchicine-binding protein may be tubulin.

scholarly journals Positive and negative regulation of basal expression of a yeast HSP70 gene.

1989 ◽  
Vol 9 (5) ◽  
pp. 2025-2033 ◽  
Author(s):  
H O Park ◽  
E A Craig
Keyword(s):  
Heat Shock ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Binding Activity ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Basal Expression ◽  
Cell Extracts ◽  
Dna Binding Assay ◽  
Kinetics Of

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.

scholarly journals Identification of concanavalin A receptors and galactose-binding proteins in purified plasma membranes of Dictyostelium discoideum.

1977 ◽  
Vol 74 (1) ◽  
pp. 264-273 ◽  
Author(s):  
C M West ◽  
D McMahon
Keyword(s):  
Dictyostelium Discoideum ◽  
Concanavalin A ◽  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Fluorescein Isothiocyanate ◽  
Binding Activity ◽  
Cellular Slime Mold ◽  
Con A ◽  
Stage Of Development ◽  
Cellular Slime

Two techniques have been modified to provide simple means for the identification of molecules which bind concanavalin A (Con A). Crossed immunoelectrophoresis was altered by replacing antibody with Con A, and receptors were identified by the precipitin arcs which they produced. Con A, tagged with fluorescein isothiocyanate, was also diffused into prefixed sodium dodecyl sulfate (SDS)-polyacrylamide gels, and additional receptors identified by fluorescence. More than 35 molecules in the plasma membranes of the cellular slime mold Dictyostelium discoideum which bind Con A were identified with these techniques. At least 12 of these diminish and 12 increase in importance as receptors during differentiation of the cells from the vegetative to the preculmination stage of development. In the course of these experiments, it was possible to confirm the presence of the galactose-binding protein discoidin, in the plasma membrane, by electrophoresing membrane proteins into an agarose gel. This lectin regains its sugar-binding activity after denaturation and electrophoresis in SDS.

Immunological detection of the oestradiol receptor protein in cell lines derived from the lymphatic system and the haematopoietic system: variability of specific hormone binding in vitro

1992 ◽  
Vol 134 (3) ◽  
pp. 397-404 ◽  
Author(s):  
F. Jakob ◽  
H. P. Tony ◽  
D. Schneider ◽  
H. H. Thole
Keyword(s):  
Cell Lines ◽  
Specific Binding ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Receptor Protein ◽  
Immunological Methods ◽  
Hormone Binding ◽  
Cell Extracts ◽  
Haematopoietic System ◽  
Mcf 7

ABSTRACT Extracts of human MCF 7 mammary carcinoma cells, the human lymphoblastoid cell lines AEH 1 and IM 9, T-cell derived CCRF cells, HL 60 myeloic leukaemia cells and murine myeloma cells SP 0 and NS I were analysed for immunoreactivity with polyclonal goat antibodies raised against homogeneous preparations of C-terminal fragments (32 kDa) of porcine uterine oestradiol receptor (ER). Whole cells and low speed cytosols were analysed for specific oestradiol-binding activity. ERs were enriched from cell extracts by either fractionated ethanol precipitation (0–25% (v/v) ethanol) and/or microscale-immunoaffinity chromatography. Immunoreactive proteins of identical molecular weight (approximately 65 kDa) were detected in all cell lines examined. Whole cell binding assays showed specific oestradiol-binding activity in MCF 7, IM 9 and CCRF cells. Borderline binding was found in HL 60 myeloid cells. No specific binding could be detected in AEH 1, NS I and SP 0 cells. Identical results were obtained using agarelectrophoresis after dextran-coated charcoal treatment. Immunoaffinity purified ERs from MCF 7, AEH 1 and HL 60 cells were subjected to limited proteolysis, where identical tryptic fragments were generated. In conclusion, we have confirmed by immunological methods that ERs are expressed in a variety of cell lines derived from the immune system and the haematopoietic system. The lack of specific hormone binding or very low-affinity hormone binding in some of the cells examined may be due to post-translational events or point mutations. Journal of Endocrinology (1992) 134, 397–404

scholarly journals Cyclic GMP binding activity in Dictyostelium discoideum

FEBS Letters ◽  
1978 ◽  
Vol 90 (2) ◽  
pp. 261-264 ◽  
Author(s):  
José M. Mato ◽  
Henry Woelders ◽  
Peter J.M. Van Haastert ◽  
Theo M. Konijn
Keyword(s):  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Binding Activity

scholarly journals Nonequilibrium kinetics of a cyclic GMP-binding protein in dictyostelium discoideum

1982 ◽  
Vol 94 (2) ◽  
pp. 271-278 ◽  
Author(s):  
PJM Van Haastert ◽  
H Van Walsum ◽  
FJ Pasveer
Keyword(s):  
Dictyostelium Discoideum ◽  
Cyclic Gmp ◽  
Binding Proteins ◽  
Binding Protein ◽  
Kinetic Constants ◽  
Nonequilibrium Conditions ◽  
Kinetics Of ◽  
Stimulation Of

Chemoattractants added to cells of the cellular slime mold dictyostelium discoideum induce a transient elevation of cyclic GMP levels, with a maximum at 10 s and a recovery of basal levels at approximately 25 s after stimulation. We analyzed the kinetics of an intracellular cGMP binding protein in vitro and in vivo. The cyclic GMP binding protein in vitro at 0 degrees C can be described by its kinetic constants K(1)=2.5 x 10(6) M(- 1)s(-1), k(-1)=3.5 x 10(-3)s(-1), K(d)=1.4 x 10(-9) M, and 3,000 binding sites/cell. In computer simulation experiments the occupancy of the cGMP binding protein was calculated under nonequilibrium conditions by making use of the kinetic constants of the binding protein and of the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions by making use of the kinetic constants of the binding protein and the shape of the cGMP accumulations. These experiments show that under nonequilibrium conditions the affinity of the binding protein for cGMP is determined by the rate constant of association (k(1)) and not by the dissociation constant (k(d)). Experiments in vivo were performed by stimulation of aggregative cells with the chemoattractant cAMP, which results in a transient cGMP accumulation. At different times after stimulation with various cAMP concentrations, the cells were homogenized and immediately thereafter the number of binding proteins which were not occupied with native cGMP were determined. The results of these experiments in vivo are in good agreement with the results of the computer experiments. This may indicate that: (a) The cGMP binding protein in vivo at 22 degrees C can be described by its kinetic constants: K(1)=4x10(6)M(-1)s(-1) and K(-1)=6x10(-3)s(-1). (b) Binding the cGMP to its binding protein is transient with a maximum at about 20-30 s after chemotactic stimulation, followed by a decay to basal levels, with a half-life of approximately 2 min. (c) The cGMP to its binding proteins get half maximally occupied at a cGMP accumulation of δ[cGMP](10)=2x10(-8) M, which corresponds to an extracellular stimulation of aggregative cells by 10(-10) M cAMP. (d) Since the mean basal cGMP concentration is approximately 2x10(-7) M, the small increase of cGMP cannot be detected accurately. Therefore the absence of a measurable cGMP accumulation does not argue against a cGMP function. (e) There may exist two compartments of cGMP: one contains almost all the cGMP of unstimulated cells, and the other contains cGMP binding proteins and the cGMP which accumulates after chemotactic stimulation. (f) From the kinetics of binding, the cellular responses to the chemoattractant can be divided into two classes: responses which can be mediated by this binding protein (such as light scattering, proton extrusion, PDE induction, and chemotaxis) and responses which cannot be (solely) mediated by this binding protein such as rlay, refractoriness, phospholipids methylation, and protein methylation.

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