Adenylate cyclase assay

Salomon, Y., Londos, C., and Rodbell, M. (1974) A highly sensitive adenylate cyclase assay. Anal. Biochem. 58, 541-548. 

Selected entries from Methods in Enzymology [vol, page(s)] Abortive complex formation, 63, 432 adenylate cyclase assay,... 

Naloxone has been synthesized 119 128) from (-)-sinomenine via (+)-7-bromodihydrocodeinone dimethyl ketal (p. 29). It possessed only 10-3-10-4 the antagonist activity of (—)-naloxone in the rat brain receptor binding assay, GPI test, and neuroblastoma x glioma hybrid cell adenylate cyclase assay. This enantiomorph may be of value to test the stereospecificity of the actions of (—)-naloxone. 

Naloxone, prepared in eight steps from (+)-7-bromodihy-drocodeinone dimethyl ketal (26% overall yield), proved virtually inactive in three test systems (binding, GPI, and neuroblastoma x glioma hybrid cell adenylate cyclase assays) and can thus be used to establish the stereospecific binding of the natural levo isomer.(37) Both antipodes of naloxone are available from a total synthesis of morphinans starting from optically active 1-benzyl-isoquinolines.(128)... 

Table 2.6 Biological Activity of Bovine PTH(l-34) and bPTH(2-34) in Rat Renal Adenylate Cyclase Assay...

In a search for more potent and selective Ai adenosine receptor agonists, and taking in mind that at Ai receptor the most active analogues were substituted adenosines, a series of N -cycloalkyl-1-deazaadenosine derivatives were synthesized and evaluated in radioligand binding studies and adenylate cyclase assays [13]. N -Cyclopentyl-2-chloro-1-deazaadenosine (1-deazaCCPA) showed the highest affinity at Ai receptor with more than two fold higher Ai selectivity than N -cyclopentyladenosine (CPA). 

Based upon differences between kidney and bone structure-activity relationships among PTH analogs, compounds should also be evaluated in a bone-based assay. Bone cell lines that contain PTH receptors such as ROS 17/2.8 cells or UMR-106 cells, are advantageous because they consist of a single bone cell type, can be maintained in culture and have also been used for both PTH binding and adenylate cyclase studies (H, ). Recently, evidence has been provided that the ROS 17/2.8 cell adenylate cyclase assay may also be used to predict weak agonist activity of PTH analogs in vivo (il.il). 

Similarly, this peptide was a partial agonist in the ROS 17/2.8 cell adenylate cyclase assay as well as the GppNHp-amplified canine renal adenylate cyclase assay (28,43). 

Measurement of adenyl cyclase activity was a laborious and imprecise procedure until the introduction of the method of Krishna et al. This method is based on the production of labeled cyclic AMP from ATP-a- P. Labeled product is separated from substrate by a combination of ion exchange chromatography and adsorption of substrate to nascent BaS04 precipitate. With appropriate equipment, it is possible to perform 300 adenyl cyclase assays in one day. A complication of the method arises from the fact that adenyl cyclase preparations invariably contain sufficient ATPase activity to deplete rapidly the substrate concentration, thereby shortening the period of time over which adenyl cyclase activity can be observed and precluding even the simplest kinetic analysis. This problem has generally been overcome by addition of an ATP-regenerating system to the adenyl cyclase assay medium . In addition, it has recently been shown that 5 -adenylyl-imidodiphosphate (AMP-PNP) is a substrate for adenyl cyclase but not for ATPase ... 

With the development of methods for plasma membrane isolation, adenyl cyclase assay, and measurement of binding of hormone to its receptor, it is now possible to study the major components of a hormone receptor. 

The complexity of the membranes and of the adenyl cyclase assay medium have forced postponement of further evaluation of the role of magnesiums and other ions. 

Table 2. Relative potencies of a-MSH and its analogs in the frog (Rana pipiens), lizard (Anolis carolinensis) skin assays, and the melanoma adenylate cyclase assay ... 

Adenylate cyclase assay was carried out into mice brain crude mitochondrial fraction by procedure based on the method of Krishna et al.(1968). 

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