Large complex formation of the inhibitor of caspase-activated DNase

Inhibitor of caspase-activated DNase (ICAD) is required for correctly folding of CAD and inhibits nuclease activity of CAD in non-apoptotic cells. From proteomic analysis of the ICAD binding proteins, we revealed that over-expressed flag-ICAD bound other ICAD molecules}. Purified recombinant ICAD protein showed three bands, 66 KDa, 132 KDa and 450 KDa, by native-PAGE. ICAD fused with glutathione-S-transferase (GST) was immunoprecipitated with anti-flag antibody from Jurkat cell lysates cotransfected with ICAD fused with either GST or flag expression vectors. When purified recombinant ICAD protein was separated by gel chromatography, the molecular weight of ICAD was detected at 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.