Summary
A sensitive method for measurement of adenylate cyclase activity in fat cell ghosts is described; it applies the protein binding assay for cyclic AMP of Gilman (1970). Unlabelled ATP is used as substrate in the presence of an ATP-regenerating system containing 5 mM creatine phosphate and 0.1 mg creatine kinase per ml. Measurement of ATP levels showed that at least 80% of the substrate level is maintained during the standard assay procedure. Chromatographic separation of cyclic AMP can be omitted, since the high specificity of the binding protein allows a dilution of the samples below the concentrations at which ATP and other nucleotides interfere with the binding of cyclic AMP. Thus, the measurement of nM concentrations of cyclic AMP in the presence of mM concentrations of ATP is achieved.
The main advantage of the method lies in the use of low protein concentrations; it reduces interfering effects of membrane-bound ATPases and phosphodiesterases. No precautions such as addition of phosphodiesterase inhibitors are needed, since cyclic AMP degradation is negligible during standard incubation conditions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bär, H. P., Hechter, O.: Adenyl cyclase assay in fat cell ghosts. Analyt. Biochem. 29, 476–489 (1969)
Birnbaumer, L., Pohl, S. L., Rodbell, M.: Adenyl cyclase in fat cells. I. Properties and the effects of adrenocorticotropin and fluoride. J. biol. Chem. 244, 3468 to 3476 (1969)
Chen, R. F.: Removal of fatty acids from serum albumin by charcoal treatment. J. biol. Chem. 242, 173–181 (1967)
Cheung, W. Y.: Inhibition of cyclic nucleotide phosphodiesterase by adenosine 5′-triphosphate and inorganic pyrophosphate. Biochem. biophys. Res. Commun. 23, 214–219 (1966)
Drummond, G. I., Duncan, L.: Adenyl cyclase in cardiac tissue. J. biol. Chem. 245, 976–983 (1970)
Ebert, R., Schwabe, U.: Biphasic effect of 5′-guanylylimidodiphosphate on fat cell adenylate cyclase. Naunyn-Schmiedeberg's Arch. Pharmacol. (in press) (1975)
Gilman, A. G.: A protein binding assay for adenosine 3′,5′-cyclic monophosphate. Proc. nat. Acad. Sci. (Wash.) 67, 305–312 (1970)
Harwood, J. P., Löw, H., Rodbell, M.: Stimulatory and inhibitory effects of guanyl nucleotides on fat cell adenylate cyclase. J. biol. Chem. 248, 6239–6245 (1973)
Hynie, S., Sharp, G. W. G.: Adenyl cyclase in the toad bladder. Biochim. biophys. Acta (Amst.) 230, 40–51 (1971)
Jakobs, K. H., Schultz, K., Schultz, G.: Hemmung von Adenyl-Cyclase-Präparationen aus der Rattenniere durch Calciumionen und verschiedene Diuretica. Naunyn-Schmiedeberg's Arch. Pharmacol. 273, 248–266 (1972)
Kalbehn, D. A., Koch, H. J.: Methodische Untersuchungen zur quantitativen Mikrobestimmung von ATP in biologischem Material mit dem Firefly-Enzymsystem. Z. klin. Chem. 5, 299–304 (1967)
Kreiner, P. W., Keirns, J. J., Bitensky, M. W.: A temperature-sensitive change in the energy of activation of hormone-stimulated hepatic adenylyl cyclase. Proc. nat. Acad. Sci. (Wash.) 70, 1785–1789 (1973)
Krishna, G., Weiss, B., Brodie, B. B.: A simple, sensitive method for the assay of adenyl cyclase. J. Pharmacol. exp. Ther. 163, 379–385 (1968)
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Maguire, M. E., Gilman, A. G.: Adenylate cyclase assay with adenylyl imidodiphosphate and product detection by competitive protein binding. Biochim. biophys. Acta (Amst.) 358, 154–163 (1974)
Pawlson, L. G., Lovell-Smith, C. J., Manganiello, V. C., Vaughan, M.: Effects of epinephrine, adrenocorticotrophic hormone, and theophylline on adenosine 3′,5′-monophosphate phosphodiesterase activity in fat cells. Proc. nat. Acad. Sci. (Wash.) 71, 1639–1642 (1974)
Pohl, S. L., Birnbaumer, L., Rodbell, M.: Glucagon-sensitive adenyl cyclase in plasma membrane of hepatic parenchymal cells. Science 164, 566–567 (1969)
Ramachandran, J.: A new simple method for separation of adenosine 3′,5′-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Analyt. Biochem. 43, 227–239 (1971)
Rodbell, M.: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. biol. Chem. 239, 375–380 (1964)
Rodbell, M., Birnbaumer, L., Pohl, S. L., Krans, H. M. J.: The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanyl nucleotides in glucagon action. J. biol. Chem. 246, 1877–1882 (1971)
Rodbell, M., Lin, M. C., Salomon, Y.: Evidence for interdependent action of glucagon and nucleotides on the hepatic adenylate cyclase system. J. biol. Chem. 249, 59–65 (1974)
Rosselin, G., Freychet, P.: Basal and hormone-stimulated adenylate cyclase in liver plasma membranes: measurement by radioimmunoassay of cyclic AMP. Biochim. biophys. Acta (Amst.) 304, 541–551 (1973)
Salomon, Y., Londos, C., Rodbell, M.: A highly sensitive adenylate cyclase assay. Analyt. Biochem. 58, 541–548 (1974)
Schönhöfer, P. S., Skidmore, I. F.: Studies on the conditions for cyclic AMP formation in homogenised and intact fat cells by use of 3H-ATP and 3H-adenine. Pharmacology 6, 109–125 (1971)
Schwabe, U., Ebert, R.: Different effects of lipolytic hormones and phosphodiesterase inhibitors on cyclic 3′,5′-AMP levels in isolated fat cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 274, 287–298 (1972)
Sheppard, H.: Inhibition of norepinephrine stimulated adenyl cyclase by theophylline. Nature (Lond.) 228, 567–568 (1970)
Steiner, A. L., Pagliara, A. S., Chase, L. R., Kipnis, D. M.: Radioimmunoassay for cyclic nucleotides. II. Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate in mammalian tissues and body fluids. J. biol. Chem. 247, 1114–1120 (1972)
White, A. A., Zenser, T. V.: Separation of cyclic 3′,5′-nucleoside monophosphate from other nucleotides on aluminum oxide columns. Application to the assay of adenyl cyclase and guanyl cyclase. Analyt. Biochem. 41, 372–396 (1971)
Yount, R. G., Ojala, D., Babcock, D.: Interaction of P-N-P and P-C-P analogs of adenosine triphosphate with heavy meromyosin, myosin, and actomyosin. Biochemistry 10, 2490–2496 (1971)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schwabe, U., Ebert, R. & Schönhöfer, P.S. Sensitive determination for adenylate cyclase activity by cyclic adenosine 3′,5′-monophosphate protein binding assay. Naunyn-Schmiedeberg's Arch. Pharmacol. 286, 83–96 (1974). https://doi.org/10.1007/BF00499106
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00499106