A Simplified Procedure for the Determination of Adenylate Cyclase Activity

Albano, J. D. M.; Maudsley, David V.; Brown, Barry L.; Barnes, Godfrey

doi:10.1042/bst0010477

Cited by 43 publications

(9 citation statements)

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“…For the determination of adenylate cyclase (AC) activity, thymocytes (2-107/ml) were lysed as in thymidine kinase assay. A mixture of 0.15 ml lysate and 0.2 ml of an assay buffer, according to Albano et al [17], consisting of MgC12 (5.25 mM), NaC1 (17.5 mM), KC1 (17.5 mM), theophylline (6 mM) and sodium ATP (3.5 mM) in Tris-HC1 (50 mM; pH 7.4), was incubated with TBTC for 10 min at 30°C. The assay was stopped with 0.35 ml of an ice-cold 24 mM sodium acetate solution (pH 4).…”

Section: Methodsmentioning

confidence: 99%

“…Adenylate cyclase activity was determined in homogenates of isolated thymocytes and varied between 50 and 90 pmol/ 107 cells per 10 rain. Sodium fluoride (5.7 mM), a known activator of the enzyme [17], was found to increase adenylate cyclase activity with a factor 4. PGE 1 (1/~M) also stimulated AC activity, namely by 40%.…”

Section: Uptake Of Glucose and Amino Acid Analogsmentioning

confidence: 99%

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Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

Snoeij

Punt

Penninks

et al. 1986

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The inhibitor of oxidative phosphorylation tri-n-butyltin chloride (TBTC) causes membrane damage and disintegration of isolated rat thymocytes at concentrations higher than 1 ixM. From a concentration of 0.1 I~M, TBTC disturbs energy metabolism as indicated by an increase in methylglucose uptake, glucose consumption and lactate production and by a decrease in cellular ATP levels. Over the same TBTC concentration range, the incorporation of DNA, RNA and protein precursors are markedly reduced. Moreover the production of cyclic AMP upon stimulation of the cells with prostaglandin E 1 is effectively inhibited. These effects cannot be explained by an inhibition of nucleoside kinase activity, amino acid uptake or adenylate cyclase activity. The effects of TBTC on macromolecular synthesis and cyclic AMP production are possibly due to a disturbance of the cellular energy state.

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Section: Methodsmentioning

confidence: 99%

Section: Uptake Of Glucose and Amino Acid Analogsmentioning

confidence: 99%

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

Snoeij

Punt

Penninks

et al. 1986

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…Purified plasma membranes stored in liquid nitrogen were used within 7 days. Adenylate cyclase activity was determined according to Albano et al (1973), evaluating the cAMP formed from unlabelled ATP (2 mM) in the presence of an ATP regeneration system (Toccafondi et al 1979). …”

Section: Kidney Cell Dispersion and Culturementioning

confidence: 99%

Parathyroid hormone bioassay using human kidney cortical cells in primary culture

Zonefrati¹,

Brandi²,

Rotella³

et al. 1982

Acta Endocrinologica

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The biological activity of parathyroid hormone (PTH) has been investigated by measuring intracellular accumulation of cyclic AMP (cAMP) in human kidney cortical cultures. Enzyme dispersed cortical cells from non-invaded kidney poles of patients undergoing nephrectomy for cancer were used after 5 days of primary culture. Bovine PTH (1\p=n-\84) produced a significant increase of cAMP accumulation in cultured cells at a dose (53.7 ng/ml) 10-fold lower than that found for the minimal stimulatory effect when using preparations of human purified plasma membranes. The action of bovine PTH (1\p=n-\84) was very rapid, a response was detected after 5 min and a ceiling effect after 30 min. Cortical cells showed a slightly lower sensitivity to synthetic bovine PTH (1\p=n-\34) (half maximal increase dose: 0.66 \g=m\g/ml),compared to bovine PTH (1\p=n-\84) (half maximal increase dose: 0.32 \g=m\g/ml),but revealed a higher sensitivity to human PTH purified from the medium of parathyroid cell cultures (half maximal increase dose: 11.2 ng/ml). Arginine-vasopressin (AVP) also increased the cAMP accumulation of kidney cortical cultured cells, with a potency and efficacy lower than that of human 'culture' PTH, while in kidney medullary cells in primary culture AVP exerted a strong response and the effect of PTH was poor or absent. Calcitonin and glucagon were weak stimulators of kidney cortical cell cAMP accumulation.

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“…Measurement ofadenylate cyclase, cyclic AMP and protein Adenylate cyclase activity of washed membrane fractions was determined by measuring the amount of cyclic AMP formed using unlabelled ATP as substrate (Albano et al, 1973). The cyclic AMP concentration in membrane and tissue slice incubates was determined in the following manner: The standard assay contained in 75 gul of final volume, 50 Sl ATP buffer (mM: 2 ATP, 3 MgCl2, 10 NaCI, 10 KCI, 2 EGTA; pH 7.4) and 25 gil assay buffer (mM: 60 Tris base, 50 HCl, 8 theophylline: pH 7.4) or drug solution in assay buffer.…”

Section: Tissue Slicesmentioning

confidence: 99%

Opioid inhibition of adenylate cyclase in the striatum and vas deferens of the rat

Bhoola

Pay

1986

British J Pharmacology

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1 The activity of adenylate cyclase in striatal membrane-enriched fractions (25,000g) was inhibited by morphine, P-endorphin, , fentanyl and bremazocine.Whereas guanosine triphosphate (GTP) appeared essential for the expression of this effect, sodium chloride seemed to enhance the degree of inhibition. Dopamine stimulation and sodium fluoride activation of the enzyme was also suppressed by morphine, ,-endorphin and DADLenk.2 P-Endorphin and DADLenk inhibited adenylate cyclase activity in vasa deferentia membraneenriched fractions (25,000 g); both opioids required GTP and NaCl and were inhibited by a 6-opioid receptor antagonist and by naloxone. Morphine, bremazocine and tifluadom did not significantly alter the activity of the vas deferens enzyme.3 Basal cyclic AMP values of striatal slices were not significantly altered by morphine, P-endorphin or DADLenk. However, dopamine-induced elevation of cyclic AMP was reduced by morphine and this effect of the opiate was suppressed by naloxone.4 Only P-endorphin lowered the basal cyclic AMP values in the vas deferens. 5 The physiological relevance of adenylate cyclase coupling to opioid receptor subtypes is considered.

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A Simplified Procedure for the Determination of Adenylate Cyclase Activity

Cited by 43 publications

References 0 publications

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

Effects of tri-n-butyltin chloride on energy metabolism, macromolecular synthesis, precursor uptake and cyclic AMP production in isolated rat thymocytes

Parathyroid hormone bioassay using human kidney cortical cells in primary culture

Opioid inhibition of adenylate cyclase in the striatum and vas deferens of the rat

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