The cyclic nucleotides, cAMP and cGMP, have been implicated as modulators of a wide variety of intracellular processes, including regulation of the cell cycle. This study was designed to monitor the levels of cAMP and cGMP at hourly intervals during the cell cycle in the syncytial plasmodium of Physarum polycephalum, and to assay the cell-cycle related activity of cAMP- and cGMP-dependent phosphodiesterase in isolated nuclei, whole-cell plasmodial homogenates, and subcellular fractions of the organism. Cyclic nucleotides were extracted in perchloric acid, washed with diethyl ether, and after removing polysaccharides with 95% ethanol, were dried and assayed by radioimmunoassay, following succinylation, using a commercially available kit. In addition to whole-cell homogenates, and sonicated isolated nuclei, the other fractions used in the phosphodiesterase assays were the supernatant and resuspended precipitate (particulate) resulting from a 10 minute, 20,000 g centrifugation of the whole-cell preparation. The phosphodiesterase activities of the various samples were assayed in an incubation mixture consisting of 50mM Tris-Hcl (pH 7.5) and 1.67mM MgCl(,2). All cell-cycle related assays, except those using isolated nuclei, were run at two final substrate concentrations, 1 x 10('-3)M and 1 x 10('-5)M for cAMP-dependent phosphodiesterase activity, and 5 x 10('-4)M and 1 x 10('-5)M for cGMP-dependent phosphodiesterase activity. Nuclear samples were assayed only at the higher substrate concentrations. Following incubation, all samples were reacted with snake venom nucleotidase to convert the generated 5' nucleotides to nucleosides. The nucleosides were separated from the cyclic nucleotide substrate (containing tracer amounts of tritiated substrate) either with fluoridated anion exchange resin, or anion exchange resin-loaded paper. Phosphodiesterase activity was calculated in terms of picomoles of cyclic nucleotide hydrolyzed per minute of incubation per milligram of protein. This study has demonstrated the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of Physarum polycephalum. In addition it has shown that with one exception the cAMP- and cGMP-dependent phosphodiesterase activity in all of the fractions fluctuates throughout the cell cycle. The whole-cell homogenate using 5 x 10('-4)M cGMP as substrate did not reveal cell-cycle related fluctuation. Kinetic data are also presented which indicate the presence of multiple phosphodiesterase activity in both the whole cell and particulate fractions for the cGMP-dependent enzyme, and in the particulate fraction for the cAMP-dependent enzyme. The derived Km values indicate that the substrate affinities of the phosphodiesterases are similar to those found previously in numerous mammalian cell studies. This study showed that cyclic nucleotides fluctuate to a significant degree during the cell cycle. No pattern was generated that was suggestive of a cause-effect relationship between the cyclic nucleotides and the cell cycle / acase@tulane.edu
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_26782 |
| Date | January 1981 |
| Contributors | Kupetz, Ira Sam (Author) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Rights | Access requires a license to the Dissertations and Theses (ProQuest) database., Copyright is in accordance with U.S. Copyright law |
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