A highly sensitive adenylate cyclase assay

Abstract

A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide. With the use of [a-"'PIATP as substrate, this method permits the nearly complete separation of cyclic ["'PIAMP formed from the substrate and other "P-containing compounds, i.e., "P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.

Constitutive Activation of Gαs within Forebrain Neurons Causes Deficits in Sensorimotor Gating Because of PKA-Dependent Decreases in cAMP

“…Temporal cortex, hippocampus, striatum, amygdala, and cerebellum were collected from either the left or right hemisphere. AC activity (basal) of membrane preparations was measured from the hippocampus and cortex in triplicate by a modification of the method by Salomon et al (1974). Aliquots (10 mg) of membrane protein were assayed in 100 ml final volume containing 0.1 mM [a-32 P]ATP (1 pCi), 2.8 U of creatine phosphokinase, 5 mM creatine phosphate, 1.5 mM MgCl 19.2% BSA, 50 mM Na-Hepes (pH 7.6), 0.3 mM KCI, 0.2 mM cAMP, and 1 mM dithiothreitol.…”

Phorbol‐ester‐induced phosphorylation of the β2‐adrenergic receptor decreases its coupling to Gs

“…Adenylyl cyclase activity was measured by the method of Satomon etal, [28] as previously described [29] using -20 #8 of membrane protein in a total volume of 0.05 ml, The incubation mixture included 0,12 mM ATP, I pCi [uJzP]ATP, 0,1 mM cAMP 0,053 mM GTP, 2,8 mM phosphoenolpyruva/e, 0,2 U of pyrt|vate kinase, I U of myokinase, 30 mM Tris-HCI (pH "7,4), 5 mM MgCl: and 0,8 mM EDTA, Enzyme activity was determined in triplicate in the absence (i.e, basal activity) or in the presence of activators (isoproterenol 0-100 ~.M, forskolin 0-100 #M), Radlo-llgand binding assays were conducted essentially as described [29] using -10 ~¢g of membrane protein in a total volume of 0,5 ml. For saturation experiments, duplicate ¢t,,,~y t~ contained ~=4Oa pM I"='IWYi ) tn the prwene= arld ~h,~r~,~ of I0 ~,M alpr~nolol, For ,/ompeth on ¢,~pcrimentt duplicate a~' tuh¢~ coat,tiled -~O pM lw:~IJCYP and 0-100 mM hopn)terenol.…”

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

“…Adenylyl cyclase activity was assayed as described by Casperson et al [23] and ['*PIcAMP produced was determined as described by Salomon et al [39] 1 ml ice-cold double-dis~led water. To measure the activity at different pH values the buffers used were as follows: sodium acetate pH 4.5-5.0, Mes pH 5.5-6.5 and Tris-NC1 pH 6.8-8.…”