Abstract
Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in Vmax as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn2+ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.
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Besch Jr HR, Jones LR, Fleming JW, Watanabe AM: Parallel unmasking of latent adenylate cyclase and Na+−K+ ATPase activities in cardiac sarcolemmal vesicles. A new use of the channel forming ionophore alamethicin. J Biol Chem 252: 7905–7908, 1977
Rothman JE, Lenard J: Membrane assymetry. Science 195: 743–753, 1977
Besch Jr HS, Jones LR, Watanabe AM: Intact vesicles of canine cardiac sarcolemma: Evidence from vectorial properties of Na+−K+ ATPase. Circ Res 39: 589, 1976
Seppet EK, Dhalla NS: Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin. Mol Cell Biochem 91: 137–147, 1989
Kato M, Kako KJ: Orientation of vesicles isolated from basolateral membranes of renal cortex. Mol Cell Biochem 78: 9–16, 1987
Pitts BJR: Stiochiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. J Biol Chem 254: 6232–6235, 1979
Hata T, Kaneko M, Beamish RE, Dhalla NS: Influence of oxygen free radicals on heart sarcolemmal Na+−Ca2+ exchange. Coronary Artery Dis 2: 397–407, 1991
Wagner JA, Reynolds IJ, Weisman HF, Dudeck P, Weisfeldt ML, Snyder SH: Ca2+ antagonists receptors in cardiomyopathic hamster: Selective increases in heart, muscle, brain. Science 232: 515–518, 1987
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with Folin phenol reagent. J Biol Chem 193: 265–275, 1951
Dixon IMC, Hata T, Dhalla NS: Sarcolemmal Na+−K+ ATPase activity in congestive heart failure due to myocardial infarction. Am J Physiol 262: C664-C671, 1992
Taussky H, Shorr E: A microcaloric method for the estimation of inorganic phosphorus. J Biol Chem 202: 678–685, 1953
Anand-Srivastava MB: Altered responsiveness of adenylate cyclase to adenosine and other agents in the myocardial sarcolemma and aorta of spontaneously-hypertensive rats. J Biochem Pharmacol 37: 3017–3022, 1988
Salmon Y, Londos C, Rodbell M: A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974
Mumby SM, Kahn RA, Manning DR, Gilman AG: Antisera of designed specifity for subunits of guanine nucleotide binding regulatory protein. Proc Natl Acad Sci USA 83: 265–269, 1986
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature London 227: 680–685, 1970
Jones L, Maddock SW, Besch Jr HR: Unmasking effect of alamethicin on the Na+−K+ ATPase, beta adrenergic receptor coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J Biol Chem 255: 9971–9980, 1980
Steinberg SF, Chow YK, Bilezikian JP: Regulation of rat heart membrane adenylate cyclase by magnesium and manganese. J Pharm Exp Ther 237: 764–772, 1986
Bonnafous JC, Dornand J, Mani JC: Detergent like effects of alamethicin on lymphocyte plasma membranes. Biochem Biophys Res Commun 86: 536–544, 1979
Downs RW, Speigel AM, Singer M, Reen S, Aurbach GD: Fluoride stimulation of adenylate cyclase is dependent on the guanine nucleotide regulatory protein. J Biol Chem 255: 949–954, 1980
Bristow MR, Ginsburg R, Strosberg A, Montgomery W, Minobe W: Pharmacology and inotropic potential of forskolin in the human heart. J Clin Invest 74: 212–223, 1984
Dixon IMC, Dhalla NS: Alterations in cardiac adrenoceptors in congestive heart failure secondary to myocardial infarction. Coronary Artery Dis 2: 805–814, 1991
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Sethi, R., Dhalla, K.S., Shah, K.R. et al. Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin. Mol Cell Biochem 119, 185–193 (1993). https://doi.org/10.1007/BF00926870
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DOI: https://doi.org/10.1007/BF00926870