scholarly journals Factors affecting the binding of [3H]adenosine 3′:5′-cyclic monophosphate to protein kinase from bovine adrenal cortex

1977 ◽  
Vol 161 (3) ◽  
pp. 653-665 ◽  
Author(s):  
S O Døskeland ◽  
P M Ueland ◽  
H J Haga
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Binding Sites ◽  
Binding Capacity ◽  
Binding Assays ◽  
Factors Affecting ◽  
Binding Characteristics ◽  
Standard Curve ◽  
Low Concentrations

Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.

scholarly journals Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

1978 ◽  
Vol 172 (3) ◽  
pp. 433-442 ◽  
Author(s):  
K. M. Jairam Menon ◽  
Salman Azhar
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Binding Capacity ◽  
Receptor Activity ◽  
Dependent Protein Kinase ◽  
Ovarian Cell ◽  
Cyclic Monophosphate ◽  
Ovarian Cells ◽  
Dependent Protein

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (Ka) and I50 (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving Ka values of 0.08 and 0.09μm and I50 of 0.12 and 0.16μm respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1μg/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (−cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1μg/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.

scholarly journals The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

1973 ◽  
Vol 136 (4) ◽  
pp. 993-998 ◽  
Author(s):  
Malcolm C. Richardson ◽  
Dennis Schulster
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Adrenocorticotrophic Hormone ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Dependent Protein

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of32P transferred from [γ-32P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1×10-2i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose–response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1×10-3i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.

EFFECT OF ENDOCRINE MANIPULATIONS ON GLUCOCORTICOID BINDING CAPACITY IN RAT SKELETAL MUSCLE

1978 ◽  
Vol 88 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Michael Mayer ◽  
Fred Rosen
Keyword(s):  
Skeletal Muscle ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Female Rats ◽  
Receptor Protein ◽  
Slight Reduction ◽  
Binding Assays ◽  
Receptor Concentration ◽  
Streptozotocin Induced Diabetes

ABSTRACT [3H]Dexamethasone binding capacity in rat muscle cytosol was determined after various endocrine manipulations in an attempt to identify factors which might regulate the level of the cytoplasmic hormone receptor protein. Hypophysectomy and adrenalectomy markedly increased the specific binding of [3H]dexamethasone in skeletal muscle cytosol, while implantation of the MtT tumour which secretes ACTH and growth hormone, as well as treatment with glucocorticoids reduced the glucocorticoid specific binding. Since the effects of hypophysectomy and the MtT tumour depend on the presence of the adrenals, they appear to be mediated via changes in circulating glucocorticoid level. Alloxan- or streptozotocin-induced diabetes caused only a slight reduction in the binding of [3H]dexamethasone in muscle, suggesting that the enhanced responsiveness to glucocorticoids in diabetes is not due to increased glucocorticoid receptor activity. There is a sex-dependent effect on binding, female rats having a higher concentration of binding sites. Furthermore, treatment with the synthetic androgen fluoxymesterone or with glucocorticoids reduces binding, while oestradiol-17β enhances it. The changes in glucocorticoid binding capacity induced by the various endocrine manipulations appear to reflect mainly changes in receptor concentration rather than occupancy, since the binding assays were preformed after a suitable time allowance for removal of the administered hormones by metabolism.

SPECIFIC BINDING OF THYROID-STIMULATING HORMONE BY HUMAN SERUM GLOBULINS

1981 ◽  
Vol 88 (3) ◽  
pp. 339-349 ◽  
Author(s):  
J. BÍRÓ
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Negative Control Group ◽  
Paper Electrophoresis ◽  
Negative Control ◽  
Thyroid Stimulating Hormone ◽  
Human Thyroid ◽  
Control Group ◽  
Binding Characteristics

Globulin preparations (41) from patients with Graves's disease (positive to thyroid stimulating immunoglobulins; TSI) and 12 from healthy persons (TSI-negative) were tested for their specific thyrotrophin (TSH)-binding properties. Globulins from both groups possessed binding sites for 131I-labelled TSH. The mean dissociation constant (Kd) was 6·8 pmol/l per mg globulin and the maximum specific binding (Bmax) was 3·0 pmol/mg globulin per 1 for the TSI-negative control group. Twenty-four (58·5%) globulin preparations from the TSI-positive group had similar TSH-binding characteristics with mean Kd of 7·2 pmol/l per mg globulin and Bmax of 3·6 pmol/mg globulin per 1 (A-type binding) but the remaining 17 (41·5%) bound TSH in a different fashion with Kd of 71·5 pmol/l per mg globulin and Bmax of 13·6 pmol/mg globulin per 1 (B-type binding). Both types of specific TSH binding reached the maximal level within 1 h of incubation and had an optimum pH of 7–8. There was a linear correlation between the amount of bound TSH and the globulin content of the samples. Both types of binding were reversible by the addition of an excess of TSH and gonadotrophins, ACTH, prolactin and insulin competed with TSH for the binding sites only when in relatively high concentrations. The binding sites were associated with macromolecules; they emerged with the void volume after chromatography on Sephadex G-200 and migrated with immunoglobulin G (IgG) on paper electrophoresis. The binding capacity of the globulin preparations could be decreased by preincubation with antiserum to human IgG or with human thyroid membranes.

scholarly journals Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

1999 ◽  
Vol 161 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Y Zhang ◽  
TA Marchant
Keyword(s):  
Binding Sites ◽  
Carassius Auratus ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Sea Bream ◽  
Single Class ◽  
Binding Characteristics ◽  
High Affinity ◽  
Goldfish Carassius Auratus ◽  
Reducing Conditions

The present study constitutes the characterization of a specific, high-affinity GH-binding protein (GHBP) in the serum of a teleost, the goldfish (Carassius auratus). GH-binding assay and ligand blotting techniques were employed to identify GHBPs in goldfish serum and hepatocyte culture medium. The binding characteristics and apparent molecular weights (Mr) of goldfish GHBPs were also compared with those of rabbit and rat. LIGAND analysis identified a single class of high-affinity and low-capacity binding sites for iodinated recombinant carp GH (rcGH) in the goldfish serum, with an association constant (Ka) of 20.1x10(9) M-1 and a maximum binding capacity (Bmax) of 161 fmol ml-1 serum. A single class of binding sites for iodinated recombinant sea bream GH and bovine GH (bGH) was also found in goldfish serum, but with a much lower affinity than that of rcGH. The binding affinity for iodinated bGH in rabbit and rat sera was found to be similar to that reported previously. Ligand blotting revealed multiple forms of GHBPs in sera of goldfish, rabbit and rat with Mr ranging from 70 kDa to 400 kDa and 27 kDa to 240 kDa under non-reducing and reducing conditions respectively. A prominent band with Mr of 66 kDa and a minor band with Mr of 27 kDa were observed to occur in sera from all three species under reducing conditions. Iodoacetamide promoted the shedding of three GHBPs with Mr of 25, 40 and 45 kDa from the cultured goldfish hepatocytes. The appearance of all bands was completely inhibited by the presence of excess unlabeled rcGH. Our results provide clear evidence that a GHBP exists in the goldfish and indicate that more information on teleost GHBPs is needed if the physiology of growth in teleosts is to be fully understood.

Triiodothyronine binding to lymphocyte nuclei and plasma cyclic AMP response to intravenous glucagon in patients with peripheral resistance to thyroid hormones

1981 ◽  
Vol 97 (1) ◽  
pp. 54-59 ◽  
Author(s):  
A. Elewaut ◽  
M. De Baets ◽  
A. Vermeulen
Keyword(s):  
Thyroid Hormone ◽  
Cyclic Amp ◽  
Binding Capacity ◽  
Peripheral Resistance ◽  
Resistance To Thyroid Hormone ◽  
Binding Characteristics ◽  
Nuclear Binding ◽  
Resistance To Thyroid Hormones ◽  
The Family

Abstract. In vitro studies of nuclear binding of triiodothyronine (T3) in lymphocytes were performed in three members of a family with hereditary peripheral resistance to thyroid hormone action. Ficoll-Hypaque® purified lymphocytes were used; the binding characteristics were analyzed by Scatchard's methods. In 5 euthyroid subjects the apparent mean equilibrium association constant (Ka) was 6.1 × 109 1/mol and the mean maximal binding capacity (Cap) 14.4 × 10−15 mol/ 100 μg DNA. In the 3 members of the family one single set of saturable T3 nuclear binding sites with affinity constants similar to those in the controls (mean Ka = 3.2 × 109 1/mol; mean Cap = 17.4 × 10−15 mol/100 μg DNA) were found. The glucagon stimulated increase in plasma cyclic AMP was studied in 6 healthy subjects and the four members of the family. The plasma cyclic AMP levels of the patients with hormone resistance were generally within the normal range. These observations demonstrate that in these patients with peripheral resistance to thyroid hormone binding of T3 to the receptor in the nucleus of lymphocytes is normal; in relation to the high circulating thyroid hormone levels, the thyroid hormone mediated cyclic AMP response is disturbed, suggesting that the defect is at the post-receptor effector level.

scholarly journals Calcineurin Inhibitor, FK506, Prevents Reduction in the Binding Capacity of Cyclic AMP-Dependent Protein Kinase in Ischemic Gerbil Brain

1997 ◽  
Vol 17 (4) ◽  
pp. 412-420 ◽  
Author(s):  
Kortaro Tanaka ◽  
Yasuo Fukuuchi ◽  
Hiroyuki Nozaki ◽  
Eiichiro Nagata ◽  
Taro Kondo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Blood Vessels ◽  
Binding Capacity ◽  
Specific Inhibitor ◽  
Artery Occlusion ◽  
Carotid Artery Occlusion ◽  
Dependent Protein Kinase ◽  
Arterial Blood ◽  
Dependent Protein

We examined the effects of FK506, a specific inhibitor of calcineurin, on the binding capacity of cyclic AMP-dependent protein kinase (cAMP-DPK) in gerbils subjected to 2-h cerebral hemispheric ischemia. FK506 (0.1 mg/kg) was infused intravenously at 15 min prior to the induction of ischemia by common carotid artery occlusion. The binding capacity of cAMP-DPK was evaluated by autoradiographic analysis of the cAMP binding, and cerebral blood flow (CBF) was measured by the [14C] iodoantipyrine method. In the sham-operated gerbils, FK506 significantly increased mean arterial blood pressure and tended to decrease CBF, suggesting that FK506 may constrict systemic blood vessels as well as cerebral blood vessels. On the other hand, cAMP binding was not altered by FK506 in the sham-operated gerbils. In the ischemia group of gerbils, FK506 prevented any significant reduction of cAMP binding in the hippocampus CA1 and cerebral cortices on the ischemic side, whereas it exerted no significant influence on the cAMP binding of the nonischemic side. The values of CBF were comparable between the vehicle-treated gerbils and FK506-treated gerbils in the ischemic regions. Preservation of cAMP binding indicates that intracellular signal transduction via cAMP-DPK can be maintained by FK506 despite ischemia, suggesting that this agent may be beneficial for reducing ischemic tissue damage.

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