Enhancement by GTP of cAMP binding to hepatic nuclei and cytosol

1984 ◽  
Vol 246 (1) ◽  
pp. C131-C140 ◽  
Author(s):  
E. M. Rosenberg ◽  
A. D. Goodman ◽  
T. L. Lipinski
Keyword(s):  
Binding Proteins ◽  
Specific Binding ◽  
Photoaffinity Labeling ◽  
Deae Cellulose ◽  
Nuclear Extract ◽  
Liver Cytosol ◽  
Nuclear Binding ◽  
Low Concentrations ◽  
Relative Molecular Weight

In the present study we have demonstrated specific binding of 3H-labeled adenosine 3',5'-cyclic monophosphate (cAMP) to a nuclear extract from rat liver. GTP, GDP, and low concentrations of ATP and ADP increased nuclear binding of [3H]cAMP, and AMP inhibited [3H]cAMP binding. Photoaffinity labeling studies employing [32P]cAMP revealed four nuclear binding proteins [relative molecular weight (Mr) 36,000, 49,000, 54,000 and 57,000]. Unlabeled cAMP decreased [32P]cAMP binding to all four proteins, whereas GTP increased binding to the 57,000 protein. We also observed specific binding of [3H]cAMP in the liver cytosol, which was stimulated by GTP but not by ADP or ATP. Photoaffinity labeling studies of the cytosol in the absence of unlabeled nucleotides revealed three cAMP-binding proteins (Mr 36,000, 49,000, and 54,000). Unlabeled cAMP inhibited binding of [32P]cAMP to all three proteins, whereas in the presence of GTP there was binding of [32P]cAMP to a Mr 57,000 protein. Using DEAE-cellulose, we isolated from the nuclear extract and cytosol a cAMP-binding protein that responded to GTP with an increase in cAMP binding but was unaffected by GDP, ATP, ADP, and AMP. Guanosine imidodiphosphate did not affect cAMP binding, suggesting that the stimulatory effect of GTP may be mediated by phosphorylation. We speculate that alterations in intracellular GTP in vivo may modulate the binding of cAMP to a protein in the nucleus and cytosol.

scholarly journals Identification of 3,5-Diiodo-l-Thyronine-Binding Proteins in Rat Liver Cytosol by Photoaffinity Labeling

Endocrinology ◽  
2003 ◽  
Vol 144 (6) ◽  
pp. 2297-2303 ◽  
Author(s):  
Maria Moreno ◽  
Elena Silvestri ◽  
Assunta Lombardi ◽  
Theo J. Visser ◽  
Fernando Goglia ◽  
...  
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Photoaffinity Labeling ◽  
Liver Cytosol ◽  
Rat Liver Cytosol

CYTOSOL AND NUCLEAR [3H]OESTRADIOL BINDING IN THE FOETAL TISSUES OF GUINEA PIG

1976 ◽  
Vol 83 (4) ◽  
pp. 811-828 ◽  
Author(s):  
J. R. Pasqualini ◽  
C. Sumida ◽  
C. Gelly
Keyword(s):  
Guinea Pig ◽  
Specific Binding ◽  
Nuclear Extract ◽  
Radioactive Material ◽  
Cytosol Fraction ◽  
Nuclear Extracts ◽  
Foetal Kidney ◽  
Foetal Lung

ABSTRACT The formation of [3H]oestradiol macromolecule complexes was studied in vivo and in vitro in the kidney, lung and liver of intact foetal guinea pig. Specific binding of [3H]oestradiol was demonstrated in the cytosol and nuclear fractions of foetal kidney. Intensive binding was found in the cytosol of foetal lung but most of the bound radioactive material (71 %) was [3H]oestrone. Some binding was found in the cytosol of foetal liver but not in the nucleus of this tissue. In the in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22 % of the plasma radioactivity was bound of which 67 % was identified as oestrone sulphate. Oestradiol sulphate represented 7 % and unconjugated oestradiol only 0.6 % (0.1 % of the total plasma radioactivity). On the other hand, 2–3 % of the foetal plasma radioactivity was found as unbound [3H]oestradiol. In the kidney, the formation of [3H]oestradiol complexes in the cytosol fraction does not depend on temperature while nuclear [3H]oestradiol complexes increase with increasing temperature. Maximal formation of [3H]oestradiol complexes in the cytosol fraction and the 0.1 m TRIS and 0.3 m NaCl nuclear extracts was reached after 15 min but binding in the 1 m NaCl nuclear extract continued to increase up to 30 min. After incubation of purified nuclei of foetal kidney with 1.1 × 10−7 m [3H]oestradiol, specific binding was found in the different nuclear fractions. Specific binding was also detected in isolated nuclei previously extracted by 0.1 m TRIS and 0.3 m NaCl before being incubated with 1.1 × 10−7 m [3H]oestradiol. The Kd of binding of [3H]oestradiol in the renal cytosol fraction is 2.5 × 10−10 m with n = 4.5 × 10−14 moles/mg protein. Incubation of isolated 1 m NaCl nuclear extract from foetal kidney with [3H]oestradiol gives a Kd of 3.3 × 10−10 m with n = 2.5 × 10−14 moles/mg protein. It is concluded that the nuclear complexes in the foetal kidney could be formed either through an intermediate cytosol complex or that the "two step" mechanism could take place in the nucleus. Furthermore, direct binding of [3H]oestradiol with high affinity was observed in the 1 m NaCl nuclear extract.

Oestradiol binding to nuclei of anterior pituitary cells of the ram

1985 ◽  
Vol 109 (1) ◽  
pp. 50-57 ◽  
Author(s):  
Marie-Lise Thieulant ◽  
Jean Pelletier
Keyword(s):  
Nuclear Receptor ◽  
Incubation Temperature ◽  
Specific Binding ◽  
Nuclear Extract ◽  
List Type ◽  
Pituitary Cells ◽  
Receptor Complex ◽  
Cytosolic Receptor ◽  
Fold Excess

Abstract. The methodology to fully characterise nuclear receptor for oestradiol-17β (E2) in the ram pituitary has been investigated. Purified nuclei, clean under the electron microscope, were obtained from 2.4 m sucrose ultracentrifugation and were extracted for 2 h at 0°C with 0.6 m NaCl. After centrifugation, the supernatant was incubated with [3H]E2 with or without a 100-fold excess of unlabelled E2. The main results were: the specific binding was maximum at 20°C in 2–3 h and remained constant up to 19 h without significant metabolism; an incubation temperature of 25°C reduces the binding, while at 0°C maximum binding was attained at a much slower rate; the binding was linearly related to the dose of nuclear proteins; the binding was not affected by DNase and RNase but was suppressed by trypsin, pronase or a temperature of 56°C; binding was specific for oestrogens; preincubation of cytosol with [3H]E2 and then coincubation with nuclei showed an uptake of the [3H]E2 receptor complex by nuclei; such a transfer was inhibited if cytosol was previously heated; after a prelabelled cytosol-nuclei coincubation, a specific binding peak was found in the nuclear extract submitted to sucrose gradient sedimentation (4.1S); in vivo injection of 100 μg E2 resulted in a sharp increase in nuclear receptor numbers 30 and 60 min later, with a concomitant drop in cytosolic receptor numbers. These results indicate that E2 can bind to pituitary nuclei in the ram.

CONCENTRATIONS OF OESTRADIOL AND OESTRONE IN PLASMA, UTERUS AND OTHER TISSUES OF FETAL GUINEA-PIGS: THEIR RELATIONSHIP TO UPTAKE AND SPECIFIC BINDING OF [3H]OESTRADIOL

1981 ◽  
Vol 89 (1) ◽  
pp. 71-77 ◽  
Author(s):  
C. GELLY ◽  
C. SUMIDA ◽  
A. GULINO ◽  
J. R. PASQUALINI
Keyword(s):  
Guinea Pigs ◽  
Specific Binding ◽  
Biological Response ◽  
Maternal Plasma ◽  
The Other ◽  
Fetal Plasma ◽  
Fetal Tissues ◽  
Low Concentrations ◽  
Selective Retention

The concentrations of unconjugated oestradiol-17β and oestrone have been measured by radioimmunoassay in the plasma of fetal, newborn and immature guinea-pigs. In fetal plasma, the values of oestradiol ranged from 15 to 50 pg/ml with no significant variations with gestational age except for an abrupt increase at the very end of gestation (148 pg/ml). Low concentrations of oestradiol were also found postnatally (from not detectable to 31 pg/ml) as well as in maternal plasma (22 pg/ml). The values of oestrone were consistently higher in all plasma regardless of age (43–164 pg/ml). Oestrogen concentrations were also determined in the fetal uterus, lung, kidney and brain and were found to be as much as 60 times higher (per g tissue) than in plasma, especially in the fetal uterus which contained four to five times more than the other tissues. These data correlated well with a 20–90 times greater uptake of [3H]oestradiol by the fetal uterus compared with the other tissues after in-vivo administration of [3H]oestradiol to the fetuses. The selective retention of oestradiol was probably due to the presence of specific oestradiol binding in these fetal tissues, particularly in the uterus whose binding was 60–120 times higher than in the other fetal tissues. Thus, the levels of oestrogen in the circulation of fetal guinea-pigs are low, but the fetal uterus is capable of maintaining a higher concentration which may be important physiologically since oestradiol has been shown to evoke a biological response in the fetal guinea-pig uterus.

In vivo Photoaffinity Labelling of Gibberellin-Binding Proteins in Avena fatua Aleurone

1993 ◽  
Vol 20 (5) ◽  
pp. 573 ◽  
Author(s):  
R Hooley ◽  
SJ Smith ◽  
MH Beale ◽  
RP Walker
Keyword(s):  
Hormonal Regulation ◽  
Binding Proteins ◽  
Specific Binding ◽  
Specific Interaction ◽  
Avena Fatua ◽  
Biologically Active ◽  
Covalent Attachment ◽  
Photoaffinity Labelling ◽  
Ga Binding Protein

It is generally accepted that specific recognition between plant hormones and proteins involved in their biosynthesis, metabolism, transport and perception are of profound importance in the hormonal regulation of plant growth and development. The identification and detailed characterisation of hormone-binding proteins which perform these functions is an important component of research that aims to understand plant hormone action. In this report the development of photoaffinity reagents for gibberellin (GA)-binding proteins is reviewed, and their use as probes with which to identify and characterise GA-binding proteins in Avena fatua aleurone is described. In vivo GA-photoaffinity labelling of aleurone layers, using the new photoaffinity probe GA4-17-sulfoxyethyl-p-azido-[125I]-iodosalicylate, leads to the covalent attachment of this reagent to numerous aleurone polypeptides. Biologically active and inactive GAs used as competitors during in vivo GA-photoaffinity labelling help discriminate between specific and non-specific binding. The biologically active GA4 and GA4-17-yl-1′- (1′-thia)propan-3′-ol-4-azido-5-iodosalicylate compete for photoaffinity labelling of a 60 kDa aleurone polypeptide, while the inactive GAs does not. These GA-photoaffinity labelling characteristics suggest that the 60 kDa aleurone polypeptide may interact specifically with active GAS. This is the first report to identify a specific GA-binding protein in aleurone. We suggest that this specific interaction observed in vivo, under conditions where the aleurone cells are responding to the GA-photoaffinity probe by expressing α-amylase genes, may be of significance in the perception and action of GA in this tissue.

Autoradiographic determination of dexamethasone binding sites along the rabbit nephron

1983 ◽  
Vol 244 (3) ◽  
pp. F325-F334 ◽  
Author(s):  
N. Farman ◽  
A. Vandewalle ◽  
J. P. Bonvalet
Keyword(s):  
Proximal Tubule ◽  
Binding Sites ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Nuclear Binding ◽  
Specific Binding Sites ◽  
In Vitro Incubation ◽  
Collecting Tubules

Specific binding sites of tritiated dexamethasone ([3H]dex) along the tubule of rabbit kidney were investigated using an autoradiographic method (dry film) on isolated tubular segments. After in vitro incubation of kidney pyramids with [3H]dex (0.15-53 nM) in the presence or absence of an excess (X200) of unlabeled dexamethasone, tubular segments were microdissected and processed for autoradiography. A quantitative analysis of specific labeling over cytoplasm and nuclei was performed. Specific nuclear binding was observed in all tubular segments beyond the pars recta. This binding was dose dependent and reached much higher values than those reported for aldosterone. In the proximal tubule, the specific labeling was also high but remained mostly cytoplasmic. The meaning of these drastically different intracellular localizations is still open to interpretation. Autoradiography was performed after in vivo injection of [3H]dex and [3H]aldosterone. The results were not different from those described here for dexamethasone and from those previously reported for aldosterone after in vitro incubation. We conclude that specific nuclear binding sites for dexamethasone range over the nephron except for proximal tubule, with no great difference among segments, in contrast to specific sites for aldosterone, which are restricted to distal and cortical collecting tubules. The exact significance of the proximal cytoplasmic specific binding of [3H]dex remains to be determined.

scholarly journals Heterogeneity and properties of hepatic dexamethasone-binding proteins

1979 ◽  
Vol 180 (1) ◽  
pp. 187-193 ◽  
Author(s):  
S L H Liu ◽  
T E Webb
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Binding Protein ◽  
Novikoff Hepatoma ◽  
Liver Cytosol ◽  
Activated Complex ◽  
Simple Dissociation ◽  
Rat Liver Cytosol

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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