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 approximately 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at approximately 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of approximately 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.
Kanouchi
H
H
Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan.
Nishizaki
H
H
Minatogawa
Y
Y
Toné
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002850:Chromatography, Gel; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D011994:Recombinant Proteins
D051017:Apoptosis Regulatory Proteins; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; C085706:FLAG peptide; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease