Determination of Ca2+/Calmodulin-Stimulated Phosphodiesterase Activity in Intact Cells
Ca2+/calmodulin (CaM)-stimulated phosphodiesterases (PDEs) constitute a large family (PDE1 family) of enzymes. All members of the PDE1 family can be stimulated by Ca2+ in the presence of CaM in vitro. It has been shown that the Ca2+/CaM-stimulated PDE activity present in the vessel wall or vascular smooth muscle cells can be stimulated in vivo by contracting reagents that increase intracellular Ca2+ concentrations. We describe in detail a technique used to estimate the extent of PDE1 activation in vivo by measuring in vitro the PDE activity that represents the extent of association between Ca2+-CaM and PDE1 in vivo. The technique involves the extraction and rapid assay of enzyme activity at a low temperature and in the presence of trifluoperazine to minimize the changes in association between Ca2+-CaM and the PDE1 family member during cell lysis and assaying activity. This technique can be used to measure Ca2+/CaM-stimulated PDE activity in cultured cells or tissues.
Key WordsCalcium calmodulin phosphodiesterase trifluoperazine intact cell
I thank James Surapisitchat for helpful comments on the manuscript. This work was supported in part by American Association Research Grant 0030302T.
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