Abstract
The association of [125I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 μM Ca2+, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 μM Ca2+. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]−1. Arrhenius-type plots of [(ro/r)-1]−1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 μM Ca2+. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F−) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Hoskins, B., Burton, C. K., Liu, D. D., Porter, A. B., and Ho, I. K. 1986. Regional and subcellular calmodulin content of rat brain. J. Neurochem. 46:303–304.
Cheung, W. Y., Bradham, L. S., Lynch, T. H., Lin, Y. M., and Tallant, E. A. 1975. Protein activator of cyclic 3′–5′-nucleotide phosphodiesterases of bovine or rat brain also activates its adenylate cyclase. Biochem. Biophys. Res. Commun. 65:1055–1062.
Olwin, B. B., and Storm, D. R. 1985. Calcium binding to complexes of calmodulin and calmodulin binding proteins. Biochemistry 24:8081–8086.
Fujisawa, H., Yamauchi, T., Nakata, H., and Okuno, S. 1984. Role of calmodulin in neurotransmitter synthesis. Fed. Proc. 43:3011–3014.
De Lorenzo, R. J. 1981. Calmodulin in neurotransmitter release and synaptic function. Fed. Proc. 41:2265–2272.
Harry, L. V., Sahyoun, N. E. 1988. Two types of brain calmodulin-dependent protein kinase Il: morphological, biochemical and immunochemical properties. Brain Res. 439:47–55.
Wang, C. L. A. 1985. A note on Ca2+ binding to calmodulin. Biochem. Biophys. Res. Commun. 130:426–430.
De Lorenzo, R. J. 1980. Role of calmodulin in neurotransmitter release and synaptic function. Ann. N. Y. Acad. Sci. 356:92–109.
Orrego, F., Riquelme, G., and Lackington, I. 1985. Interaction of calmodulin antagonists with plasma membrane and with plasma membrane lipids. In: Calmodulin antagonists and cellular physiology. pp. 321–333, Acad. Press.
Asakawa, T., Takano, M., Enomoto, K., and Hayama, K. 1984. Ca2+-activated, fatty acid-dependent guanylate cyclase in synaptic plasma membranes and its requirement for Ca2+ and Mg-GTP in the activation. Adv. Exp. Med. Biol. 175:173–186.
Bunn, S. J., Garthwaite, J., and Wilkin, G. P. 1986. Guanylate cyclase activities in enriched preparations of neurones, astroglia and synaptic complex isolated from rat cerebellum. Neurochem. Int. 8:179–185.
Kopeikina-Tsiboukidou, L., and Deliconstantinos, G. 1986. Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes. Int. J. Biochem. 18:777–784.
Papaphilis, A., and Deliconstantions, G. 1980. Modulation of serotonergic receptors by exogenous cholesterol in the dog brain synaptosomal plasma membranes. Biochem. Pharmacol. 29:3325–3327.
Miller, G. L. 1959. Protein determination for large numbers of samples. Analyt. Chem. 31:964–968.
Ellman, G. L., Courteny, D., Andres, V., and Featherstone, R. M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88–95.
Shinitzky, M., and Barenholz, Y. 1978. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochem. Biophys. Acta 515:367–394.
Deliconstantinos, G. 1986. Prostaglandin F2a binding on dog brain synaptosomal plasma membranes and its evoked effects on membrane fluidity, (Na++K+)-stimulated ATPase and Ca2+-stimulated ATPase activities. Cell Mol. Biol. 32:113–119.
Deliconstantions, G., Kopeikina-Tsiboukidou, L., and Villiotou, V. 1987. Evaluation of membrane fluidity effects and enzyme activities alterations in adriamycin neurotoxicity. Biochem. Pharmacol. 36:1153–1161.
Scatchard, G. 1949. The attractions of protein for small molecules and ions. Ann. N. Y. Acad. Sci. 51:660–672.
Mc Murchie, E. J., and Raison, J. K. 1979. Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes. Biochem. Biophys. Acta 554:364–374.
Silman, I., and Futerman, S. H. 1987. Modes of attachment of acetylcholinesterase to the surface membrane. Eur. J. Biochem. 170:11–22.
Deliconstantinos, G. 1983. Phenobarbital modulates the (Na++K+)-stimulated ATPase and Ca2+-stimulated ATPase activities by increasing the bilayer fluidity of dog brain synaptosomal plasma membranes. Neurochem. Res. 8:1143–1152.
Farias, N. F. 1987. Insulin-membrane interactions and membrane fluidity changes. Biochem. Biophys. Acta 906:459–468.
Deliconstantions, D. 1988. Structure activity relationship of cholesterol and steroid hormones with respect to their effects on the Ca2+-stimulated ATPase and lipid fluidity of synaptosomal plasma membranes from dog and rabbit brain. Comp. Biochem. Physiol. 89B:585–594.
Deliconstantinos, G., Daefler, S., and Krueger, G. R. F. 1987. Cholesterol modulation of membrane fluidity and ecto-nucleotide triphosphatase activity in human and CLL lymphocytes. Anticancer Res. 7:347–352.
Cheung, W. V. 1982. Calmodulin: an overview. Fed. Proc. 41:2253–2257.
Graf, E., Filoteo, A. G., and Penniston, J. T. 1981. Association of125I-calmodulin with retention of full biological activity: its binding to human erythrocyte ghosts. Arch. Biochem. Biophys. 203:719–726.
Grinstein, S., and Furuya, W. 1982. Binding of125I-calmodulin to platelet a-granules. FEBS Lett. 140:49–52.
Tsakiris, S., and Deliconstantinos, G. 1984. Influence of phosphatidylserine on (Na++K+)-stimulated ATPase and acetylcholinesterase activities of dog brain synaptosomal plasma membranes. Biochem. J. 220:301–307.
Brasitus, T. A., Dudeja, P. K. 1986. Modulation of lipid fluidity of small and large-intestinal antipodal membranes by Ca2+. Biochem. J. 239:652–661.
Deliconstantinos, G., and Tsakiris, S. 1985. Differential effect of anionic and cationic drugs on the synaptosomal-associated acetylcholinesterase activity. Biochem. J. 229:81–86.
Sweet, W. D., Wood, W. G., Schroeder, F. 1987. Charged anesthetics selectively alter plasma membrane order. Biochemistry 26:2828–2835.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lioudmila, KT., Deliconstantinos, G. Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes. Neurochem Res 14, 119–127 (1989). https://doi.org/10.1007/BF00969626
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00969626