Separation of cyclic 3′,5′-nucleoside monophosphates from other nucleotides on aluminum oxide columns application to the assay of adenyl cyclase and guanyl cyclase

Brain and caudate nucleus adenylate cyclase: Effects of dopamine, GTP, E prostaglandins and morphine

“…After incubating the samples at 30°C for 5 to 20 min, the reaction was stopped by boiling the samples for 2 min. The labelled CAMP formed was purified by chromatography on alumina columns (23); the results were verified by isolation of CAMP by Dowex (50W X 4) chromatography followed by a double Ba(OH)aZn SO4 precipitation [24] . 3 H-Naloxone binding studies were performed as previously described [3].…”

“…We did not measure the CAMP formed by the method of Gilman [25] but used instead labelled ATP ( [(Y-~' P] ATP, 5 X 1 O6 cpm per assay) which was synthesized as described by Symons [26,27] and purified (> 95% ATP) by paper chromatography (Bennett and Cuatrecasas, unpublished). After incubating the samples at 30°C for 2.5 min, the reaction was stopped in a boiling water bath for 2 min; 1 ml of [8-3H] CAMP (30 000 cpm) was added to each tube to allow calculations of recovery, and the 32P-CAMP formed was purified by chromatography on alumina [23]. Over 95% of the 32P-product formed was identified as CAMP by DEAE column chromatography (Siegel and Cuatrecasas, unpublished).…”

Calcium ion: A modulator of parotid adenylate cyclase activity

“…Reactions were terminated by the addition of 0.1 ml 40 mM Tris-HCl (pH 7.5), 40 mM ATP and 12.5 mM cAMP 3H (10 000 cpm). cAMP was isolated on columns of neutral alumina [6] with a recovery of 90-95%. The reaction rate was linear with time and with protein concentration under the assay conditions used.…”

Modulation of Adenylate Cyclase/Cyclic AMP Response by Thyrotropin and Prostaglandin E2 in Cultured Thyroid Cells. 1. Negative Regulation

“…The pellets were solubilized in NaOH for protein determinations. The supernatants were applied to alumina columns to purify cyclic AMP [9]. Aliquots of the cyclic AMP fractions were diluted appropriately and used for assay of cyclic AMP by the competitive protein binding assay of Gilman [lo] with minor modifications [ll].…”