Separation and assay of phosphodiesterase isoforms in murine peritoneal macrophages using membrane matrix DEAE chromatography and [32P]cAMP.
Cyclic nucleotide phosphodiesterases (PDE) are a family of exquisitely regulated enzymes which play a central role in regulating the biological half-life of cAMP and cGMP. Hormonal regulation of specific isoforms is the basis for crosstalk and antagonism between several well-described signaling pathways. In the present work improved methods are described which accelerate and simplify the separation and assay of PDE isoforms occurring in mouse peritoneal macrophages. The described method is equally applicable to other cell and tissue types, and is based on the mobilization of DEAE beads on a macroporous support structure which allows high flow rates, high resolution, and reproducible separations. The failure of the resin to undergo compression in this configuration prevents band spreading caused by diffusion into small pores and channeling associated with bed movement. Therefore, the PDE isoforms from a tissue extract can be resolved in a chromatographic run taking only 30-35 min. In addition, the column cartridge can be regenerated and fully reequilibrated in 10-15 min. After each chromatographic run, cAMP PDE activity in the fractions is characterized by incubating each fraction with [32P]cAMP, followed by quantitative conversion of [32P]AMP formed in the initial reaction to 32PO4 and adenosine. The unreacted [32P]-cAMP is then adsorbed onto charcoal and after centrifugation, the 32PO4 remaining in the supernatant is determined by counting an aliquot. The latter step replaces the need to use small columns to separate tritiated cAMP from tritiated adenosine as in previous assays, and provides the expected improvement in sensitivity associated with a high specific activity substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Uhing
R J
RJ
Gettys
T W
TW
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D000242:Cyclic AMP / Q000138:chemical synthesis; D005069:Evaluation Studies as Topic; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010761:Phosphorus Radioisotopes
D007527:Isoenzymes; D010761:Phosphorus Radioisotopes; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases