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- Identification of Potassium-Dependent and -Independent Components: RESULTS(6)

The direct suppressive effects of these ions could be overcome, however, because all three ions tested were unable to suppress plasmid DNA degradation catalyzed by high amounts of nuclease (0.1 U/reaction; Fig. 9C; data not shown for 0.1 U DNase-I) whereas SAM remained an effective inhibitor of nuclease activity (Fig. 9C). The ability of 150 mM KCl to inhibit plasmid DNA degradation catalyzed by increasing amounts of DNase-I or DNase-II is shown in Figure 9, B and D, respectively.

Modulation of Caspase Activity by KCl

Caspases are believed to be central to the execution of apoptosis in a wide variety of cell types, including germ cells and granulosa cells of the ovary. Moreover, these enzymes are believed to function upstream of DNA cleavage, with release of caspase-activated DNase from its inhibitor protein offered as an example. Therefore, we next analyzed the sensitivity of recombinant caspases to inhibition by potassium and other ions using cell-free proteolysis assays, as previously described. Cleavage of pro-interleukin (IL)-1p to its mature form is a major function for caspase-1 and a clear indicator of its activity. As shown in Figure 10A, incubation of pro-IL1p with 1 ^g of recombinant caspase-1 resulted in rapid cleavage of the protein to the mature cytokine. The presence of 150 mM KCl, NaCl, or LiCl in the assay mixture inhibited caspase-1-mediated cleavage of pro-IL1p, although under these experimental conditions, the extent of inhibition was not complete because first-site cleavage was observed in all cases (Fig. 10A). However, cleavage of pro-IL1p by lower concentrations of caspase-1 (100 ng) was completely suppressed by all of the ions tested (data not shown). Similarly, we observed that the ability of 1 ^g of recombinant cas-pase-3 to catalyze cleavage of cytoplasmic actin was inhibited by the presence of 150 mM KCl, NaCl, or LiCl (Fig. 10B).
Fig10Identification of Potassium
FIG. 10. Potassium-mediated suppression of caspase activity in cell-free substrate cleavage assays. A) Inhibitory effects of 150 mM KCl, NaCl, or LiCl on the sequential processing of recombinant pro-IL1 p (33 kDa) to the mature cytokine (17 kDa) catalyzed by 1 ^g of recombinant caspase-1. B) Inhibition of caspase-3-mediated cleavage of actin (intact protein, 42 kDa; first cleavage product, 41 kDa), present in crude cytoplasmic extracts prepared from rat granulosa cells, by 150 mM KCl, NaCl, or LiCl.

October 13, 2013 Cells
Tags: apoptosis follicle granulosa cells ovum