scholarly journals Isolation, characterization and distribution of adenosine 3′:5′-cyclic monophosphate from Pinus radiata

1978 ◽  
Vol 175 (3) ◽  
pp. 931-936 ◽  
Author(s):  
T Wilson ◽  
E Moustafa ◽  
A G C Renwick
Keyword(s):  
Statistical Analysis ◽  
Cyclic Amp ◽  
Pinus Radiata ◽  
Degrees Of Freedom ◽  
Binding Assay ◽  
Gel Filtration ◽  
Plant Tissues ◽  
Competitive Binding Assay ◽  
Cyclic Monophosphate ◽  
Solvent Systems

Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.

The Effect of Aging on β-Adrenoceptor-Stimulated Cyclic AMP Formation in Human Lymphocytes

1981 ◽  
Vol 60 (5) ◽  
pp. 587-589 ◽  
Author(s):  
C. A. Kraft ◽  
C. M. Castleden
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Binding Assay ◽  
Human Lymphocytes ◽  
Phosphodiesterase Inhibitor ◽  
Adenylate Cyclase System ◽  
Competitive Binding Assay ◽  
Cyclic Monophosphate ◽  
Maximal Stimulation ◽  
Stimulation Of

1. Responsiveness of the β-adrenoceptor adenylate cyclase system was measured in lymphocytes from healthy young and old subjects by incubating the cells with isoprenaline in the presence of a phosphodiesterase inhibitor and by measuring production of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) with a competitive binding assay. 2. The two groups did not differ significantly in the levels of cyclic AMP produced or in the concentration of isoprenaline required to give half-maximal stimulation of the cells (ED50).

scholarly journals Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation

1987 ◽  
Vol 245 (3) ◽  
pp. 923-924 ◽  
Author(s):  
T A Santa-Coloma ◽  
M A Bley ◽  
E H Charreau
Keyword(s):  
Cyclic Amp ◽  
Protein Binding ◽  
Ammonium Sulphate ◽  
Cyclic Nucleotide ◽  
Half Life ◽  
Binding Assay ◽  
Great Stability ◽  
Competitive Binding Assay ◽  
Ammonium Sulphate Precipitation ◽  
Protein Binding Assay

The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.

scholarly journals The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release. Properties of islet-cell adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1972 ◽  
Vol 129 (4) ◽  
pp. 945-952 ◽  
Author(s):  
D. J. Sams ◽  
W. Montague
Keyword(s):  
Cyclic Amp ◽  
Insulin Release ◽  
Islets Of Langerhans ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Cyclic Monophosphate ◽  
Km Value

1. An assay has been developed with sufficient sensitivity for determination of the adenosine 3′:5′-cyclic monophosphate diesterase activity in islets of Langerhans, and has been used to investigate the response of the enzyme to various agents which are known to affect insulin release. 2. The subcellular distribution of the enzyme in islets of Langerhans prepared from guinea-pig pancreas was investigated and over 70% of the activity present in the original homogenate was recovered in the supernatant fraction. 3. Gel filtration of the activity present in the supernatant fraction on Sephadex G-200 gave a single peak of activity with an apparent molecular weight of 200000. The phosphodiesterase activity in the peak fraction showed two apparent Km values for adenosine 3′:5′-cyclic monophosphate (cyclic AMP) of 3μm and 30μm, suggesting the presence of two activities. The pH optimum of the activity with the low Km value was 8.7. 4. Theophylline, caffeine, 3-isobutyl-1-methylxanthine (SC-2964), glibenclamide, tolbutamide, xylitol and leucine were inhibitors of the activity with the low Km value; imidazole and arginine stimulated the activity, and glucose and diazoxide were without significant effect. 5. It is suggested that the agents theophylline, caffeine, SC-2964, glibenclamide, tolbutamide, leucine and imidazole may alter the intracellular concentration of cyclic AMP in islets of Langerhans by affecting the cyclic AMP phosphodiesterase activity in islet cells and in this way may affect insulin release.

scholarly journals Studies of the active site of m-calpain and the interaction with calpastatin

1993 ◽  
Vol 296 (1) ◽  
pp. 135-142 ◽  
Author(s):  
C Crawford ◽  
N R Brown ◽  
A C Willis
Keyword(s):  
Ion Exchange ◽  
Proteolytic Activity ◽  
Active Site ◽  
Binding Assay ◽  
Large Subunit ◽  
Gel Filtration ◽  
Small Subunit ◽  
Binding Domain ◽  
Competitive Binding Assay ◽  
Binding Domains

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

The Absorption, Clearance and Metabolic Fate of Dermatan Sulphate Administered to Man - Studies Using a Radioiodinated Derivative

1989 ◽  
Vol 62 (03) ◽  
pp. 945-949 ◽  
Author(s):  
J Dawes ◽  
B A Hodson ◽  
D S Pepper
Keyword(s):  
Binding Assay ◽  
Degradation Products ◽  
Gel Filtration ◽  
Dermatan Sulphate ◽  
Metabolic Fate ◽  
Healthy Human ◽  
Competitive Binding Assay ◽  
Human Volunteers ◽  
Highly Correlated ◽  
Dose Dependent

SummaryAn iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.

Aptamer-Based Enantioselective Competitive Binding Assay for the Trace Enantiomer Detection

2007 ◽  
Vol 79 (12) ◽  
pp. 4716-4719 ◽  
Author(s):  
Josephine Ruta ◽  
Corinne Ravelet ◽  
Isabelle Baussanne ◽  
Jean-Luc Décout ◽  
Eric Peyrin
Keyword(s):  
Binding Assay ◽  
Competitive Binding ◽  
Competitive Binding Assay

An improved calibration curve between soil pH measured in waterand CaCl2

Soil Research ◽  
2002 ◽  
Vol 40 (8) ◽  
pp. 1399 ◽  
Author(s):  
B. L. Henderson ◽  
E. N. Bui
Keyword(s):  
Statistical Analysis ◽  
Water Resources ◽  
Soil Ph ◽  
Calibration Curve ◽  
Smooth Function ◽  
Degrees Of Freedom ◽  
Additive Model ◽  
Smoothing Spline ◽  
Look Up Table ◽  
6 Degrees Of Freedom

A new pH water to pH CaCl2 calibration curve was derived from data pooled from 2 National Land and Water Resources Audit projects. A total of 70465 observations with both pH in water and pH in CaCl2 were available for statistical analysis. An additive model for pH in CaCl2 was fitted from a smooth function of pH in water created by a smoothing spline with 6 degrees of freedom. This model appeared stable outside the range of the data and performed well (R2 = 96.2, s = 0.24). The additive model for conversion of pHw to pHCa is sigmoidal over the range of pH 2.5 to 10.5 and is similar in shape to earlier models. Using this new model, a look-up table for converting pHw to pHCa was created.

scholarly journals Intracellular calcium and adenosine 3′,5′-cyclic monophosphate as mediators of potassium-induced aldosterone secretion

1985 ◽  
Vol 228 (1) ◽  
pp. 69-76 ◽  
Author(s):  
I Kojima ◽  
K Kojima ◽  
H Rasmussen
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Intracellular Calcium ◽  
Secretory Response ◽  
Ionophore A23187 ◽  
Small Decrease ◽  
Aldosterone Secretion ◽  
Cyclic Monophosphate ◽  
A Value ◽  
Glomerulosa Cells

We compared the action of K+ on aldosterone secretion from isolated bovine adrenal glomerulosa cells with that of ionophore A23187. Addition of either 50 nM-A23187 or 8 mM-K+ to perifused cells induces a similar initial aldosterone-secretory responses, and a similar sustained increases in Ca2+ entry. However, K+-induced secretion is more sustained than is A23187-induced secretion, even though each agonist appears to act by increasing Ca2+ entry into the cells. When [3H]inositol-labelled cells are stimulated by 8 mM-K+, a small decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is observed. This decrease is not accompanied by an increase in inositol trisphosphate (InsP3) concentration. Also, if [3H]arachidonic acid-labelled cells are exposed to 8 mM-K+, there is no increase in [3H]diacylglycerol production. When [3H]inositol-labelled cells are stimulated by 50 nM-A23187, a small decrease in PtdIns(4,5)P2 is observed. This decrease is not accompanied by an increase in InsP3. The cyclic AMP content of K+-treated cells was approximately twice that in A23187-treated cells. If cells are perifused simultaneously with 50 nM-forskolin and 50 nM-A23187, the initial aldosterone-secretory response is similar to that induced by A23187 alone, and the response is sustained rather than transient, and is similar to that seen during perifusion of cells with 8 mM-K+. This dose of forskolin (50 nM) causes an elevation of cyclic AMP concentration in A23187-treated cells, to a value similar to that in K+-treated cells. These results indicate that, in K+-treated cells, a rise in cyclic AMP content serves as a positive sensitivity modulator of the Ca2+ message, and plays a key role in mediating the sustained aldosterone-secretory response.

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

1973 ◽  
Vol 12 (11) ◽  
pp. 2683-2685 ◽  
Author(s):  
Eric G. Brown ◽  
Russel P. Newton
Keyword(s):  
Plant Tissues ◽  
Cyclic Monophosphate
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