Cyclic AMP phosphodiesterase activity in the nucleus of the myxomycete Physarum flavicomum was demonstrated by cytochemical staining utilizing electron microscopy and by enzymatic assays with tritiated cyclic AMP as the substrate. Cytochemical staining showed Physarum's plasmodial phosphodiesterase activity to be located in the nucleus, along the plasma membrane, in vesicles, and free in the cytoplasm. Nuclear phosphodiesterase, which may be cell cycle dependent, was primarily located in the nucleolus. Nuclei from three to five day old microplasmodial cultures were isolated by the method of Henney and Yee. Whole cells were collected through centrifugation and washed. Pellets were homogenized in a medium composed of 0.01 MTris-HC1 (pH 7.2 at 4 °C), 0.25 M sucrose, 0.01% Triton X-100, and 5mM CaC1₂. Nuclei were collected through double filtration and two 1.0 M sucrose density gradient centrifugations. After the nuclei were washed, microscopic examination revealed a purity of over 90%. Radioactive assays of the nuclear preparations demonstrated phosphodiesterase activity consistant with that indicated by cytochemical localization. The specific activity of the nuclear enzyme was 15 nMole of cyclic AMP hydrolyzed /min/mg. of protein.
Bean, Judith A.; Kleve, Maurice G.; and Lynch, Thomas J.
"Isolation of Nuclei from Physarum flavicomum: Demonstration of Nuclear Cyclic Acid AMP Phosphodiesterase,"
Journal of the Arkansas Academy of Science: Vol. 40
, Article 6.
Available at: http://scholarworks.uark.edu/jaas/vol40/iss1/6